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Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the αβ bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(kcat/KM) for the reaction correlated well with the Hammett σ- parameter with ρ = −0.39 ± 0.03; r² = 0.93. The negative ρ value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin-product adduct through a cyclo-elimination reaction. [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP, amylin) forms pancreatic amyloid in type-2 diabetes, a process that contributes to the loss of β-cell mass in the disease. IAPP has been found in all higher organisms examined, but not all species form amyloid and the ability to do so correlates with the primary sequence. The amyloidogenic potential of fish IAPPs has not been examined, although fish have been proposed as a source for xenobiotic transplantation. The sequence of pufferfish IAPP (Takifugu rubripes) is known and is the most divergent from human IAPP of any reported IAPP sequence, differing at 11 positions including seven located within residues 20-29, a segment of the molecule that is important for controlling amyloidogenicity. Several of the substitutions found in pufferfish IAPP are nonconservative including Ser to Pro, Asn to Thr, Ala to Tyr, and Leu to Tyr replacements, and several of these have not been reported in mammalian IAPP sequences. Amyloid prediction programs give conflicting results for pufferfish IAPP. CD spectroscopy, FTIR, and transmission electron microscopy reveal that pufferfish IAPP forms amyloid and does so more rapidly than human IAPP in tris buffer at pH 7.4, but does so more slowly in phosphate buffered saline (PBS) at pH 7.4. Molecular dynamics simulations indicate that the pufferfish sequence is compatible with models of IAPP amyloid. The fish polypeptide does not significantly bind to thioflavin-T in tris and does so only weakly in PBS. The results highlight difficulties with thioflavin-T assays and the ambiguity in defining amyloidogenicity. [ABSTRACT FROM AUTHOR]

Zinc is reported to play a complex role in islet amyloid polypeptide (IAPP) aggregation, which is associated with β-cell death in type II diabetes (T2D). Depending on their relative concentrations in vitro, zinc could either promote or inhibit IAPP aggregation. Interestingly, genomewide association studies suggested both positive and negative correlations between T2D risks and activities of a β-cell-specific zinc transporter upon mutations, which determines zinc concentration in vivo. To decipher the effect of zinc coordination on IAPP aggregation, we performed atomistic discrete molecular dynamics simulations to systemically study aggregation propensities of zinc-coordinated IAPP oligomers with different molecular weights (MWs), whose populations are determined by zinc concentration. We find that at low zinc:IAPP stoichiometry, zinc coordination promotes aggregation by forming high-MW oligomers. The aggregation is inhibited when the stoichiometry increases and zinc binds individual peptides. Our computationally derived predictions are validated by the complementary thioflavin-T fluorescence assay measuring the dependence of IAPP aggregation on a wide range of zinc concentrations. Our combined computational and experimental study offers detailed mechanistic insight into the complex role of zinc on IAPP aggregation and T2D development. [ABSTRACT FROM AUTHOR]

In probing the oxygen reactivity of an Enterococcus faecalis NADH oxidase (Nox; O2 → 2H2O) C42S mutant lacking the Cys42-sulfenic acid (Cys42-SOH) redox center, we provided direct evidence of a C(4a)-peroxyflavin intermediate in the oxidative half-reaction and also described a conformational or chemical change that is rate-limiting for full reoxidation of the homodimer. In this work, the Nox from Streptococcus pyogenes (SpyNox) has been expressed and crystallized, and the overoxidized wild-type [Cys44-SOH → Cys44-sulfinic acid (Cys44-SO2H)] and C44S mutant enzyme structures have been refined at 2.0 and 2.15 Å, respectively. We show that azide binds to the two-electron reduced wild-type (EH2) enzyme and to the mutant enzyme in solution, but with a significantly higher affinity for the mutant protein. The spectral course of the titration with the SpyNox EH2 form clearly indicates progressive displacement of the Cys44-S- → FAD charge-transfer interaction. An azide soak with C44S Nox crystals led to the structure of the complex, as refined at 2.10 Å. The active-site N3- ligand is proximal to the Ser44 and His11 side chains, and a significant shift in the Ser44 side chain also appears. This provides an attractive explanation for the azide-induced loss of charge-transfer absorbance seen with the wild-type EH2 form and also permits accommodation of a C(4a)-peroxyflavin structural model. The conformation of Ser44 and the associated helical element, and the resulting steric accommodation, appear to be linked to the conformational change described in the E. faecalis C42S Nox oxidative half-reaction. [ABSTRACT FROM AUTHOR]

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Enzyme-catalyzed reactions often rely on a noncovalently bound cofactor whose reactivity is tuned by its immediate environment. Flavin cofactors, the most versatile catalyst encountered in biology, are often maintained within the protein throughout numbers of complex ionic and aromatic interactions. Here, we have investigated the role of π-π stacking and hydrogen bond interactions between a tyrosine and the isoalloxazine moiety of the flavin adenine dinucleotide (FAD) in an FAD-dependent RNA methyltransferase. Combining several static and time-resolved spectroscopies as well as biochemical approaches, we showed that aromatic stacking is assisted by a hydrogen bond between the phenol group and the amide of an adjacent active site loop. A mechanism of recognition and binding of the redox cofactor is proposed. [ABSTRACT FROM AUTHOR]

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-β (Aβ) from Alzheimer's disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aβ fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aβ and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aβ and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution. [ABSTRACT FROM AUTHOR]

Aureochromes have been shown to act as blue-light-regulated transcription factors in algae in the absence of phototropins. Aureochromes comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module binding the flavin chromophore and a basic region leucine zipper (bZIP) domain as an effector. The domain arrangement in aureochromes with an N-terminal effector is inversed to other LOV proteins. To clarify the role of the linking A'α helix in signaling, we have investigated the LOV domain of aureochrome1a from the diatom alga Phaeodactylum tricornutum without the N-terminal A'α helix but with the C-terminal Jα helix. Results were analyzed in comparison to those previously obtained on the LOV domain with both flanking helices and on the LOV domain with the A'α helix but without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence by a band at 1656 cm-1 that the A'α helix unfolds in response to light. This unfolding takes place only in the presence and as a consequence of the unfolding of the Jα helix, which points to an allosteric regulation. Size exclusion chromatography shows the LOV domain to be dimeric in the absence and monomeric in the presence of the A'α helix, implying that the folded helix covers the dimerization site. Therefore, the A'α helix directly modulates the oligomerization state of the LOV domain, whereas the Jα helix acts as an allosteric regulator. Both the allosteric control and the light-induced dimerization have not been observed in phototropin-LOV2 and point to a different signaling mechanism within the full-length proteins. [ABSTRACT FROM AUTHOR]

The misfolding, aggregation, and accumulation of proteins as amyloid fibrils is a defining characteristic of several debilitating diseases. Human apolipoprotein C-II (apoC-II) amyloid fibrils are representative of the fibrils formed by a number of plasma apolipoproteins implicated in amyloid-related disease. Previous structural analyses identified a buried charge pair between residues K30 and D69 within apoC-II amyloid fibrils. We have investigated the effects of amino acid substitutions of these residues on apoC-II fibril formation. Two point mutations of apoC-II, D69K and K30D, as well as a reversed ion-pair mutant containing both mutations (KDDK) were generated. Fibril formation by the double mutant, apoC-II KDDK, and apoC-II D69K was enhanced compared to that of wild-type (WT) apoC-II, while apoC-II K30D lacked the ability to form fibrils under standard conditions. Structural analyses showed that WT apoC-II, apoC-II D69K, and apoC-II KDDK fibrils have similar secondary structures and morphologies. Size distribution analyses revealed that apoC-II D69K fibrils have a broader range of fibril sizes while apoC-II KDDK fibrils showed an increased frequency of closed fibrillar loops. ApoC-II D69K fibrils exhibited reduced thioflavin T binding capacity compared to that of fibrils formed by WT apoC-II and apoC-II KDDK. These results indicate that specific charge and charge-pair mutations within apoC-II significantly alter the ability to form fibrils and that position 69 within apoC-II plays a key role in the rate-limiting step of apoC-II fibril formation. [ABSTRACT FROM AUTHOR]

Barnacles are unique marine sessile crustaceans and permanently attach to various foreign surfaces during most of their lifespan. The protein complex secreted from their body and used to attach their calcareous shell to almost all surfaces in water has long fascinated us because we have limited technology with which to attach materials in water. Unraveling the mechanism of underwater attachment by barnacles is thus important for interface science, for the understanding of the biology and physiology of barnacles, and for the development of technology to prevent fouling. Previous studies have indicated that the intermolecular interactions optimized by conformations of the adhesive proteins are crucial in the self-assembly and/or curing of the adhesive. This study aimed to identify the possible structural determinants responsible for the self-assembly. Thioflavin T binding screening of peptides designed on the basis of the primary structure of a bulk 52 kDa cement protein indicated the presence of some amyloidogenic motifs in the protein. The conformation of the peptide was transformed to a β-sheet by an increase in either pH or ionic strength, resulting in its self-assembly. Thioflavin T binding was inhibited by small polyphenolic molecules, suggesting the contribution of aromatic interactions during self-assembly. The occurrence of amyloid-like units in the protein implies that the protein conformation is an important factor contributing to the self-assembly of the cement, the first event of the curing, as the adhesive material is secreted into the seawater out of the animal's body. [ABSTRACT FROM AUTHOR]

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of Lornithine, L-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD (P) H-dependent N-hydroxylase known as Ktzl. We have been able to structurally characterize Ktzl in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavindependent hydroxylases and adds another layer to the complexity of flavoenzymes. [ABSTRACT FROM AUTHOR]

Flavo-diiron proteins (FDPs) function as anaerobic nitric oxide scavengers in some microorganisms, catalyzing reduction of nitric to nitrous oxide. The FDP from Thermotoga maritima can be prepared in a deflavinated form with an intact diferric site (deflavo-FDP). Hayashi et al. [(2010) Biochemistry 49, 7040-7049] reported that reaction of NO with reduced deflavo-FDP produced substoichiometric N2O. Here we report a multispectroscopic approach to identify the iron species in the reactions of deflavo-FDP with NO. Mossbauer spectroscopy identified two distinct ferrous species after reduction of the antiferromagnetically coupled diferric site. Approximately 60 % of the total ferrous iron was assigned to a diferrous species associated with the N2O-generating pathway. This pathway proceeds through successive diferrous-mononitrosyl (S = 1/2 FeII{FeNO}7) and diferrous-dinitrosyl (S = 0 [{FeNO}7]2) species that form within ~ 100 ms of mixing of the reduced protein with NO. The diferrous-dinitrosyl intermediate converted to an antiferromagnetically coupled diferric species that was spectroscopically indistinguishable from that in the starting deflavinated protein. These diiron species closely resembled those reported for the flavinated FDP [Caranto et al. (2014) J. Am. Chem. Soc. 136, 7 981- 7992], and the time scales of their formation and decay were consistent with the steady state turnover o f the flavinated protein. The remaining ~ 40% of ferrous iron was inactive in N2O generation but reversibly bound NO to give an S = 3/2 {FeNO}7 species. The results demonstrate that N2O formation in FDPs can occur via conversion o f S = 0 [{FeNO}7]2 to a diferric form without participation of the flavin cofactor. [ABSTRACT FROM AUTHOR]

Continuous-flow mass spectrometry (CFMS) was used to monitor the products formed during the initial 0.25-20 s of the reactions catalyzed by the flavoprotein N- acetylpolyamine oxidase (PAO) and the pterin-dependent enzymes phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH). N,N'-Dibenzyl-1,4-diaminobutane (DBDB) is a substrate for PAO for which amine oxidation is rate-limiting. CFMS of the reaction showed formation of an initial imine due to oxidation of an exo-carbon-nitrogen bond. Nonenzymatic hydrolysis of the imine formed benzaldehyde and N-benzyl-1,4-diaminobutane; the subsequent oxidation by PAO of the latter to an additional imine could also be followed. Measurement of the deuterium kinetic isotope effect on DBDB oxidation by CFMS yielded a value of 7.6 ± 0.3, in good agreement with a value of 6.7 ± 0.6 from steady-state kinetic analyses. In the PheH reaction, the transient formation of the 4a-hydroxypterin product was readily detected; tandem mass spectrometry confirmed attachment of the oxygen to C(4a). With wild-type TyrH, the 4a-hydroxypterin was also the product In contrast, no product other than a dihydropterin could be detected in the reaction of the mutant protein E332A TyrH. [ABSTRACT FROM AUTHOR]

Amyloid deposits in vivo are complex mixtures composed of protein fibrils and nonfibrillar components, including polysaccharides of the glycosaminoglycan (GAG) class. It has been widely documented that GAGs influence the initiation and progress of self-assembly by several disease-associated amyloidogenic proteins and peptides in vitro. Here we investigated whether the GAG heparin can serve as a cofactor to induce amyloid-like fibril formation in a peptide predicted to have a weak propensity to aggregate and not associated with amyloid disorders. We selected the 23-residue peptide PLB(1–23), which corresponds to the acetylated cytoplasmic domain of the phospholamban transmembrane protein. PLB(1–23) remains unfolded in aqueous solution for >24 h and does not bind thioflavin T over this time period, in agreement with computer predictions that the peptide has a very low intrinsic amyloidogenicity. In the presence of low-molecular mass (5 kDa) heparin, which binds PLB(1–23) with micromolar affinity, the peptide undergoes spontaneous and rapid assembly into amyloid-like fibrils, the effect being more pronounced at pH 5.5 than at pH 7.4. At the lower pH, peptide aggregation is accompanied by a transition to a β-sheet rich structure. These results are consistent with the polyanionic heparin serving as a scaffold to enhance aggregation by aligning the peptide molecules in the correct orientation and with the appropriate periodicity. PLB(1–23) is toxic to cells when added in isolation, and promotion of fibril formation by heparin can reduce the toxicity of this peptide, consistent with the notion that amyloid-like fibrils represent a benign end stage of fibrillization. This work provides insight into the role that heparin and other glycosaminoglycans may play in amyloid formation and provides therapeutic avenues targeting the reduction of cytotoxicity of species along the amyloid formation pathway. [ABSTRACT FROM AUTHOR]

TrmFO is a tRNA methyltransfera.se that uses methylenetetrahydrofo-late (CH2THF) and flavin adenine dinucleotide hydroquinone as cofactors. We have recently shown that TrmFO from Bacillus subtilis stabilizes a TrmFO-CH2-FADH adduct and an ill-defined neutral flavin radical. The adduct contains a unique N-CH2-S moiety, with a methylene group bridging N-5 of the isoalloxazine ring and the sulfur of an active-site cysteine (Cys53). In the absence of tRNA substrate, this species is remarkably stable but becomes catalytically competent for tRNA methylation following tRNA addition using the methylene group as the source of methyl. Here, we demonstrate that this dormant methylating agent can be activated at low pH, and we propose that this process is triggered upon tRNA addition. The reaction proceeds via protonation of Cys53, cleavage of the C-S bond, and generation of a highly reactive [FADH(N5)CH2]' iminium intermediate, which is proposed to be the actual tRNA-methylating agent. This mechanism is fully supported by DFT calculations. The radical present in TrmFO is characterized here by optical and EPR/ENDOR spectroscopy approaches together with DFT calculations and is shown to be the one-electron oxidized product of the TrmFO-CH2-FADH adduct. It is also relatively stable, and its decomposition is tacilitated by high pH. These results provide new insights into the structure and reactivity of the unique flavin-dependent methylating agent used by this class of enzymes. [ABSTRACT FROM AUTHOR]

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putidaS12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FADox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s-1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium-binding studies indicate that NSMOA binds FADhq 13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein-protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD+ after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions. [ABSTRACT FROM AUTHOR]

Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell penetration and low cytotoxicity of fibril probes having been widdely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadiuplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT. Here, we investigated the binding mechanism of ThT with several variants of the human telomeric sequence in the presence of monovalent cations. Fluorescence titrations and electrospray ionization mass spectrometry (ESl-MS) analyses demonstrated that each G-quadruplex unit cooperatively binds to several ThT molecules. ThT brightly fluoresces when a single ligand is bound to the G-quadruplex and is quenched as ligand binding stoichiometry increases. Both the light-up signal and the dissociation constants are exquisitely sensitive to the base sequence and to the G-quadrupIex structure. These results are crudal for the sensible design and interpretation of G-quadruplex detection assays using fluorescent ligands in general and ThT in particular. [ABSTRACT FROM AUTHOR]

PixD is a blue light-using flavin (BLUF) photoreceptor that controls phototaxis in the cyanobacterium Synechocystis sp. PCC6803. PixD interacts with the response regulator-like protein PixE in a light-dependent manner, and this interaction is critical for light signal transduction in vivo. However, the structure of the PixD-PixE complex has not been determined. To improve our understanding of how PixD transmits its captured light signal to PixE, we used blue-native polyacrylamide gel electrophoresis to characterize the molecular mass of a recombinant PixD-PixE complex purified from Escherichia coli and found it to be 342 kDa, suggesting that the complex contains 10 PixD and 4 PixE monomers. The stoichiometry of the complex was confirmed by Western blotting. Specifically, three intermediate states, PixD10-PixE1, PixD10-PixE2, and PixD10-PixE3, were detected. The apparent dissociation constant for PixE and PixD is ∼5 μM. A docking simulation was performed using a modeled PixE structure and the PixD10 crystal structure. The docking simulation showed how the molecules in the PixD10-PixE4 structure interact. To verify the accuracy of the docked model, a site-directed mutagenesis study was performed in which Arg80 of PixE, which appears to be capable of interacting electrostatically with Asp135 of PixD in the predicted structure, was shown to be critical for complex formation as mutation of PixE Arg80 to Asp or Ala prevented PixD-PixE complex formation. This study provides a structural basis for future investigations of the light signal transduction mechanism involving PixD and PixE. [ABSTRACT FROM AUTHOR]

The oxidation of the reduced flavin in choline oxidase was investigated with pH, solvent viscosity, and kinetic isotope effects (KIEs) in steady-state kinetics and time-resolved absorbance spectroscopy of the oxidative half-reaction in a stopped-flow spectrophotometer. Both the effects of isotopic substitution on the KIEs and the multiple KIEs suggest a mechanism for flavin oxidation in which the H atom from the reduced flavin and a H+ from the solvent or a solvent exchangeable site are transferred in the same kinetic step. Stopped-flow kinetic data demonstrate flavin oxidation without stabilization of flavin-derived species. Solvent viscosity effects establish an isomerization of the reduced enzyme. These results allow us to rule out mechanisms for flavin oxidation in which C4a-peroxy and -hydroperoxy flavin intermediates accumulate to detectable levels in the reaction of flavin oxidation catalyzed by choline oxidase. A mechanism of flavin oxidation that directly results in the formation of oxidized flavin and hydrogen peroxide without stabilization of reaction intermediates is consistent with the data presented. [ABSTRACT FROM AUTHOR]

Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings. [ABSTRACT FROM AUTHOR]

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ1-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition. [ABSTRACT FROM AUTHOR]

The first demonstrated example of a regulatory function for a bacterial hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O2 and undergo autoxidation. The amino acid sequence encoded by the VC1216 gene from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial diguanylate cyclases (DGCs) that catalyze formation of cyclic di-(3',5')-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon being exposed to air. Vc Bhr-DGC showed approximately 10 times higher DGC activity in the diferrous than in the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in V. cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low-dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity. [ABSTRACT FROM AUTHOR]

Oligomenc forms of β-amyloid (Aβ) have potent neurotoxic activity and are the primary cause of neuronal injury and cell death in Alzheimer's disease (AD). Compounds that perturb oligomer formation or structure may therefore be therapeutic for AD. We previously reported that D.[(chGly)-(Tyr)-(chGly)- (chGly)-(mLeu)]-NH2 (SEN304) is able to inhibit Aβ aggregation and toxicity, shown primarily by thioflavin T fluorescence and MTT (Kokkoni, N. et al. (2006) N-Methylated peptide inhibitors of β-amyloid aggregation and toxicity. Optinlisation of inhibitor structure. Biochemistry 45, 9906-9918). Here we extensively characterize how SEN304 affects Aβ(1-42) aggregation and toxicity, using biophysical assays (thioflavin T, circular dichroism, SDS-PAGE, size exdusion chromatography, surface plasmon resonance, traveling wave ion mobility mass toxicity assays in cell culture (MTT and lactate dehydrogenase in human SH-SHYSY cells, mouse neuronal cell death and synaptophysin) and long-term potentiation in a rat hippocampal brain slice. These data, with dose response curves, show that SEN304 is a powerful inhibitor of Afl( 1-42) toxicity, particularly effective at preventing Al) inhibition of long-term potentiation. It can bind directly to Aβ(1-42), delay β-sheet formation and promote aggregation of toxic oligomers into a nontoxic form, with a different morphology that cannot bind thioflavin T. SEN304 appears to work by inducing aggregation, and hence removal, of Aβ oligomers. It is therefore a promising lead compound for Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the kcat/Km(Al) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O2 constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH- to O2, without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H+ proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H2O2. No solvent KIE was found for O2 reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on kcat/Km(Ox), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations. [ABSTRACT FROM AUTHOR]

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2═O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential. [ABSTRACT FROM AUTHOR]

Vibrio harveyi NADPH-FMN oxidoreductase (FRP) catalyzes flavin reduction by NADPH. In comparing amino acid sequence and crystal structure with Escherichia coli NfsA, residues N134, R22S, Rl33, K167, and R15 were targeted for investigation of their possible roles in the binding and utilization of the NADPH substrate. By mutation of each of these five residues to an alanine, steady-state rate analyses showed that the variants K167A and R1SA had apparently greatly increased Km, NADPH and reduced kcat/Km,NADPH, whereas little or much more modest changes were found for the other variants. The deuterium isotope effects D(V/K) for (4R)-[4-2H]-NADPH were markedly increased to 6.3 and 7.4 for K167A and R1SA, respectively, indicating that the rate constants for NADPH and NADP+ dissociation were greatly enhanced relative to the hydride transfer steps. Also, anaerobic stopped-flow analyses revealed that the equilibrium dissociation constant for NADPH binding (Kd) to be 2.5-3.9 and 1.1 mM for K167A and R1SA, respectively, much higher than the 0.4 μM Kd for the native FRP, whereas the kcat of these two variants were similar to that of the wild-type enzyme. Moreover, the K167 to alanine mutation led to even a slight increase in kcat/Km for NADH. These results, taken together, provide a strong support to the conclusion that K167 and R15 each was critical in the binding of NADPH by FRP. Such a functional role may also exist for other FRP homologous proteins. [ABSTRACT FROM AUTHOR]

The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA. [ABSTRACT FROM AUTHOR]

In nntochondria, complex I (NADH quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron- sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-duster chain extending from the flavin) are (at least partially) reduced by NADH. The other three dusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyrne~ they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use Mtissbauer spectroscopy on 57Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent dusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential dusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential dusters. The implications for intramolecular electron transfer along the extended chain of cthctors in complex I are discussed. [ABSTRACT FROM AUTHOR]

Typically, simple fiavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen per-oxide without release of significant levels of the superonde ion. However, two evolutionarily related single-domain sulihydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predom-inantly found in the mitochondrial intermembrane) release up to ∼30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofzctor within the helix-rich Erv domain. Subsequent reduction of the fiavin is followed by transfer of reduc-ing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (TI-TP) as an alter-native reducing substrate to dithiothreitol (DTT). Till', and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the fonnation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent ampli-fication of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemilusninescence method using a luciferin analogue (MCLA) when 2 mM DTI' was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~-~6S% when mono-meric mutants of the normally homodimeric enzyme were used. In contrast~ monomeric multidomain quiescin sulihydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed. [ABSTRACT FROM AUTHOR]

Mycobacterium smegmatis G (MbsG) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of the terminal amino group on the side chain of l-lysine in the biosynthetic pathway of the siderophore mycobactin. Mycobactins are essential for mycobacterium growth under iron-limiting conditions encountered during infection in mammals. Thus, enzymes involved in the biosynthesis of mycobactin represent potential drug targets. MbsG was expressed in Escherichia coli and purified using metal affinity and ionic exchange chromatographies. Recombinant MbsG represents the first member of this class of enzymes isolated in the active form, with a tightly bound FAD cofactor. The kcat value for formation of hydroxylated l-lysine under steady-state conditions was 5.0 min-1, and Km values of 0.21 mM for l-lysine, 1.1 mM for NADH, and 2.4 mM for NADPH were calculated. The enzyme functioned as an oxidase when the activity of MbsG was measured by monitoring oxygen consumption in the absence of l-lysine, oxidizing NADH and NADPH with kcat values of 59 and 49 min-1, respectively. Under these conditions, MbsG produced both hydrogen peroxide and superoxide. In contrast, when l-lysine was present, the reaction became more coupled, producing hydroxylated l-lysine and decreasing the oxidase activity. These results suggest that substrate binding modulates the function of MbsG from an oxidase to a monooxygenase [ABSTRACT FROM AUTHOR]

Fructosamine oxidases (FAOXs) are flavin-containing enzymes that catalyze the oxidative deglycation of low molecular weight fructosamines or Amadori products. The fructosamine substrate is oxidized by the flavin in the reductive half-reaction, and the reduced flavin is then oxidized by molecular oxygen in the oxidative half-reaction. The crystal structure of FAOX-II from Aspergillus fumigatus reveals a unique interaction between Lys53 and the isoalloxazine. The ammonium nitrogen of the lysine is in contact with and nearly centered over the aromatic ring of the flavin on the si-face. Here, we investigate the importance of this unique interaction on the reactions catalyzed by FAOX by studying both half-reactions of the wild-type and Lys53 mutant enzymes. The positive charge of Lys53 is critical for flavin reduction but plays very little role in the reaction with molecular oxygen. The conservative mutation of Lys53 to arginine had minor effects on catalysis. However, removing the charge by replacing Lys53 with methionine caused more than a million-fold decrease in flavin reduction, while only slowing the oxygen reaction by ~30-fold. [ABSTRACT FROM AUTHOR]

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower kcat/Km value (0.04 ± 0.01 μM-1 min-1) relative to wild-type (1.17 ± 0.22 μM-1 min-1), there was no activity observed with the R297A SsuD variant. Each of the arginine variants had similar Kd values for flavin binding as wild-type SsuD (0.32 ± 0.15 μM), but there was no measurable binding of octanesulfonate. The low levels of activity for the R297A and R297K SsuD variants correlated with the absence of any detectable C4a-(peroxy)flavin formation in stopped-flow kinetic studies. Single-turnover experiments were performed in the presence of SsuE to evaluate both the reductive and oxidative half-reaction. With wild-type SsuD a lag phase is observed following the reductive half-reaction by SsuE that represents flavin transfer or conformational changes associated with the binding of substrates. Evaluation of the Arg297 SsuD variants in the presence of SsuE showed no lag phase following reduction by SsuE, and the flavin was oxidized immediately following the reductive half-reaction. These results corresponded with a lack of detectable changes in the proteolytic susceptibility of R297A and R297K SsuD in the presence of reduced flavin and/or octanesulfonate, signifying the absence of a conformational change in these variants with the substitution of Arg297. [ABSTRACT FROM AUTHOR]

PixD (Slr1694) is a BLUF (blue-light-using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study, we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light-excited state and only minimally accelerates its decay from 5.7 to 4.5 s. However, mutations of Tyr8, Gln50, and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher-order PixD10-PixE5 complex, which is indicative of a lit conformational state. Solution nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo-light-excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild-type PixD. [ABSTRACT FROM AUTHOR]

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these poiar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX · chloride complex requires Lys26S and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX chloride complex and a ternary MSOX · chloride · MTA complex Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four hgands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe ofpolar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities. [ABSTRACT FROM AUTHOR]

Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (Kd = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression. [ABSTRACT FROM AUTHOR]

The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (APPABLUF) reconstituted with isotopically labeled riboflavin (RI) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics. [ABSTRACT FROM AUTHOR]

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide a-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer. [ABSTRACT FROM AUTHOR]

The AppA BLIJF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue, Gln63, that is thought to undergo a shift in hydrogen-bonding interactions when a bound flavin is light excited. In this study we have characterized two substitution mutants ofGln63 (Q63E, Q63L) in the context of two Constructs of the BLUE domain that have differing lengths, AppA1-126and AppA 17-133. Q63L mutations in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle. Altered fluorescence emission and fluorescence quenching of the Q63L mutant indicate significant perurbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed by ¹H-15N HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signaling state as evidenced by a permanent 3 am red shift of the flavin absorption, quenching of flavin fluorescence emission, analysis of ¹H-15N HSQC spectra, and the inability of full-length AppA Q63E to bind to the PpsR repressor. The significance of these findings on the mechanism of light-induced output signaling is discussed. [ABSTRACT FROM AUTHOR]

Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air- sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active Site. Alternative mechanisms for the nitric oxide reductase (NOR) activity consisting of either protonation ofa diiron-hridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal FMNH2 in the rate-determining step have been proposed. To test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron Site but does not exhibit multiturnover NOR or O2 reductase (O2R) activity. Reactions of the reduced (diferrous) deulavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to one NO per diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = ⅛ EPR signal arising from antiferromagnetic coupling of an S = ⅜ (FeNO)7 with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N2O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O2R activities upon simple addition of FMN. The production of N2O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo- FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron- mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs. [ABSTRACT FROM AUTHOR]

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillusfu,niga:us under iron-limiting conditions. This siderophore contains N5-hydroxylated L-ornithines essential for iron binding. A.fumigaius siderophore A (AJSidA) catalyzes the flavin- and NADPH-dependent hydroxylation of L-ornithine in ferrichrome biosynthesis. AJSidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to L-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. AJSidA is highly specific for its amino acid substrate, only hydroxylating L-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, A! SidA can be reduced by NADPFI, and a C4a-(hydro)- peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by L-Ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP in the stabilization of intermediates in the reaction of AJSidA. NADP1 is a competitive inhibitor with respect to NADPH, demonstrating that AfSidA forms a ternary complex with NADP+ and L-ornithine during catalysis. The data suggest that AfSidA likely proceeds by a sequential kinetic mechanism. [ABSTRACT FROM AUTHOR]

The su1fhdryI oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short. cytokine-like. form of ALR (5fALR), and a longer form (IfALR) which resides in the mitochondrial interniembrane space (IMS). IfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia4O during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi. T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni. A., Fortunato, F., Nizzardo, M., Napoli. L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I.. and Comi, G. p. (2009) Am. J. Hu,n. Gene!. 84, 594-6041 described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human RI94H mutant in If- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR. close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2'-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10°C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NM R spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al. [ABSTRACT FROM AUTHOR]

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N³ atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H2O2 as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N³-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by D-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H2O2 D-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases ~22-fold compared to that of the wild-type enzyme. The reduction of H167A using D-glucose isotopes (2-d-D-glucose, 3-d-D-glucose, and 1,2,3,4,5,6, 6-d7-D-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of D-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the D-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the D-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters. [ABSTRACT FROM AUTHOR]

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR 1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 Å resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code IF6M). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Set72 in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification. [ABSTRACT FROM AUTHOR]

The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants ≥ 105 M-1 s-1 and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is < 6 A from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by ∼50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases. [ABSTRACT FROM AUTHOR]

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudontonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 Å resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA ∼8000 limes more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with Ihe other. A much weaker ∼2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with it preferred restction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction. [ABSTRACT FROM AUTHOR]

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved ice helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-Jα and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the ice linker helix was observed in LOV1-LOV2-Jα in agreement with previous studies of LOV2-Jα. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the ice linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in heicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process. [ABSTRACT FROM AUTHOR]

Proline utilization A (PutA) from Escherichia coil is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to its enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ϵ-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline-reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin State to the distal membrane-binding domain include major reorganization of the flavin ribityl chain, severe (35°) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5) atom, Arg43I, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-α1 loop to complete the active site. [ABSTRACT FROM AUTHOR]

There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudotnomtas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumnannü enzyme, an example of the alternative group. The reductase and oxygenase from P. aeruginosa were expressed in Escherichia coil. The reductase was purified as a stable C-terminally Histagged yellow protein containing weakly bound FAD, and the oxygenase was purified as a stable colorless N-terminally His-tagged protein. The reductase catalyzes the reduction of FAD by NADH and releases the FADH product into solution, but unlike the reductase from A. bau,nannii, this catalysis is not influenced by HPA. The oxygenase binds the released FADH and catalyzes the oxygenation of HPA to form 3,4dihydroxyphenylacetate, after which the FAD dissociates to be re-reduced by the reductase, a common overall pattern for two-component flavin-dependent oxygenases. With this system, it appears that interactions between the reductase and the oxygenase can facillitate the transfer of FADH to the oxygenase, although they are not required. We show that the P. aeruginosa oxygenase system in complex with FADH reacts with 02 to form a quasi-stable, unusually high-extinction flavin hydroperoxide species that binds HPA and reacts to form the product. The resultant flavin hydroxide decomposes to FAD and water while still bound to the oxygenase and then releases product and FAD from the protein. Unlike the enzyme from A. baumannii, during normal catalysis involving both the reductase and oxygenase, the rate-determining step in catalysis is the dissociation of FAD from the oxygenase in a process that is independent of the concentration of HPA. Structures for the reductases and oxygenases from A. bau,nannii and from Thermus the rmophilus (similar to the P. aeruginosa system) form a basis for interpreting the molecular origins of the differences between the two groups of flavin-dependent two-component oxygenases. [ABSTRACT FROM AUTHOR]

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine lO-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis niduians and from Escherichia coil was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH). Flashinduced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. [ABSTRACT FROM AUTHOR]

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory chain enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. Alternatively, NADH oxidation, by the flavin mononucleotide in complex I, can be coupled to the reduction of hydrophilic electron acceptors, in non-energy-transducing reactions. The reduction of molecular oxygen and hydrophilic quinones leads to the production of reactive oxygen species, the reduction of nicotinamide nucleotides leads to transhydrogenation, and "artificial" electron acceptors are widely used to study the mechanism of NADH oxidation. Here, we use a combined modeling strategy to accurately describe data from three flavin-linked electron acceptors (molecular oxygen, APAD+, and ferricyanide), in the presence and absence of a competitive inhibitor, ADP-ribose. Our combined ping-pong (or ping-pong-pong) mechanism comprises the Michaelis-Menten equation for the reactions of NADH and APAD+, simple dissociation constants for nonproductive nucleotide-enzyme complexes (defined for specific flavin oxidation states), and second-order rate constants for the reactions of ferricyanide and oxygen. The NADH-dependent parameters are independent of the identity of the electron acceptor. In contrast, a further flavin-linked acceptor, hexaammineruthenium(III), does not obey ping-pong-pong kinetics, and alternative sites for its reaction are discussed. Our analysis provides kinetic and thermodynamic information about the reactions of the flavin active site in complex I that is relevant to understanding the physiologically relevant mechanisms of NADH oxidation and superoxide formation. [ABSTRACT FROM AUTHOR]