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Reports on the coryneform bacterium, Brevibacterium ammoniagenes, which contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B. Construction of specific fasA and fasB expression systems for the isolation of homogenous preparations of both enzymes; Finding that the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence; Differences in the ability of the B ammoniogenes type I fatty acid synthases to synthesize oleic acid and in their chain-length specificities.

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity: The enzyme appeared to be encapsulated in particles obtained alter mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25 % (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acre 309, 11–22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case. [ABSTRACT FROM AUTHOR]

Arg44, located at the si-face side of the flaving ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-um resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The Φ, Ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis. [ABSTRACT FROM AUTHOR]

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase- FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 ≈ 20 h at 0 °C in air: the Arrhenius ΔHdecay= being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction. [ABSTRACT FROM AUTHOR]

By sulfur substitution of the N-5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5-thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half-reduced states, but not in the oxidized state. Among the three 'redox shuttles' characterizing the flavin system, viz. upper 1e-, lower 1e- and 2e- shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e- transfer. The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a '2e- flavin', 5-deazaflavin, has been treated in the preceding paper. 5-Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the 'blue' flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5-thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer. Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical. The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred + Fe(CN)63- ⇄ sFl• + Fe(CN)64-, the standard potential of the sFlred/sFl• couple, Em at pH 7, has been estimated as + 0.38 V. [ABSTRACT FROM AUTHOR]

An extracellular enzyme which utilizes molecular oxygen to oxidize cellodextrins to the corresponding aldonic acids has been isolated from culture filtrates of the white-rot fungus Sporotrichum puh,erulentum. This enzyme, tentatively named cellobiose oxidase, has been highly purified by classical techniques and has been demonstrated to be a glycoprotein with a molecular weight of approximately 93000. Ultraviolet spectra of the enzyme in the presence and absence of substrate are characteristic of a hemoprotein. Acidic hydrolyses of the enzyme followed by a spectrofluorimetric investigation of the hydrolysate has demonstrated the presence of approximately one flavin component per enzyme molecule. The possible role of this complex enzyme in cellulose degradation is discussed. [ABSTRACT FROM AUTHOR]

1. The addition of Ca2+ to purified nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa results in a 2–8-fold increase in flavin fluorescence, closely resembling that induced by 2′-AMP (adenosine 2′-monophosphate). The response to Ca2+ is sigmoidal, with a Hill-coefficient of 2.6. Reduction of thionicotinamide adenine dinucleotide (oxidised form) by NADH responds in a similar fashion to Ca2+ and 2′-AMP. 2. Mg2+ inhibits the Ca2+-dependent activation in a competitive fashion, whereas no effect of Mg2+ is observed when the enzyme is activated with 2′-AMP. 3. The pH dependencies of the activations by 2′-AMP and Ca2+ are different. The activation by Ca2+ shows little or no dependence on pH, whereas higher concentrations of 2′-AMP are required to obtain the same activation when the pH is increased. 4. The addition of both Ca2+ and 2′-AMP causes a dramatic decrease in the concentration of Ca2+ required for half-maximal stimulation. 5. A negative cooperative effect of NADP+ has been demonstrated indirectly, i.e. addition of NADP+ increases the concentration of Ca2+ required for half-maximal stimulation. 6. These results are interpreted as indicating the existence of separate allosteric binding sites for Ca2+ and 2′-AMP. Furthermore, Ca2+ is proposed to act as a heterotropic ligand. The results are discussed in terms of current allosteric models. [ABSTRACT FROM AUTHOR]

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactor dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, and analogue computer simulation of the dissociation as well as manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein. [ABSTRACT FROM AUTHOR]

1. Initial velocity studies on β-cyclopiazonate oxidocyclase in the presence of the competitive inhibitor, 3-acetyl-5-scatyl-tetramic acid, indicates that this enzyme catalyzes the oxidocyclization of β-cyclopiazonic acid by a Ping-Pong-Bi-Bi mechanism. 2. It was possible to reduce the flavin coenzyme to the 3,4-dihydroflavin by consecutive cycles of reduction with BH4- and reoxidation by atmospheric oxygen. 8. The BH4-reduced enzyme shows a similar affinity for indole derivatives as does the native flavoquinone enzyme, but is inactive. β-Cyclopiazonic acid binds to the enzyme in a simple equilibrium, and the reduction of the 3,4-dihydroflavin to the 1,3,4,5.tetrahydroflavin or a conversion to 1,5-dihydroflavin does not proceed. 4. Partial reactivation of the enzyme by the photocatalyzed oxidation of the 3,4-dihydroflavin to the flavoquinone form was observed [ABSTRACT FROM AUTHOR]

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the μtant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (ε[submax] = 11400 M[sup-1] cm[sup-1]). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F μtant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme (Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619). This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the μtant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F μtant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the μtant of 0.74 s[sup-1] versus 20 s[sup-1] for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave K[subm] values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The K[subm] for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the K[subm] for oxygen is μch lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s[sup-1]. [ABSTRACT FROM AUTHOR]

Human glutathione reductase (NADPH + GSSG + H+ ⇄ NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD > 8-azido-FAD = 8-F-FAD = 8-Cl-FAD > 4-thio-FAD = l-deaza-FAD > 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD · glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focused on position 8 of the flavin. 8-Cl-FAD-glutathione reductase and 8-F-FAD · glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD · glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. [ABSTRACT FROM AUTHOR]

The reaction of 2-hydroxy-3-butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393–399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5. Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate-inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798–1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself. Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate, it is proposed that differences in the rate-determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyaeid oxidase. [ABSTRACT FROM AUTHOR]

3,4-Pentadienoyl-CoA, an allenic substrate analog, is a potent inhibitor of the flavoprotein pig-kidney general acyl-CoA dehydrogenase. The analog reacts very rapidly (k = 2.4 × 103 min-1) with the native oxidized enzyme to form a covalent flavin adduct probably involving tile isoalloxazine position N-5. This species is inactive, but activity may be regained by two pathways. The allenic thioester can be displaced (k = 0.3 min-1) by a large excess of octanoyl-CoA substrate upon reversal of covalent adduct formation. Alternatively, tile enzyme inactivator adduct slowly decomposes (t½ = 75 min) to form the strongly thermodynamically favoured 2,4-diene and catalytically active, oxidized enzyme. During this latter process 15–20% of the activity is irreversibly lost probably due to covalent modification of the protein. These data suggest that 3,4-pentadicnoyl-CoA should be considered a suicide substrate of the acyl-CoA dehydrogenase. The mechanism of the reactions, and in particular the 3,4→2,4 tautomerization, are consistent with a catalytic sequence initiated by abstraction of an α-hydrogen as a proton. [ABSTRACT FROM AUTHOR]

The redox properties of the covalently-bound flavin and of the tetrahedral iron-sulfur center S1 of succinate dehydrogenase were studied as a function of the binding of different ligands to the enzyme. The midpoint potential of both flavin and S1 increases by some 200 mV when protein binds succinate to a site having Ksuccd = 0.8-1.0 mM, thus different from the substrate binding site. Succinate binding increases the potential of the oxidized flavin/semiquinone half-cell more than that of the semiquinone/reduced flavin one : this results in higher semiquinone formation with increasing succinate. Malonate and fumarate appear to mimic, in this regard, the effect of succinate. The increase in midpoint potential of S1 upon binding of dicarboxylic acid is related to an increase in hydrophobicity of the cluster environment. The possible molecular basis for the modulation of the flavin potential is discussed together with the significance of this shift on the catalytic behaviour of the protein. [ABSTRACT FROM AUTHOR]

The natural flavin of laetate oxidase, FMN, was removed and replaced by the synthetic flavin 2-thioriboflavin 5'- phosphate (2-thio FMN). Despite the differences in properties of the flavins, including an oxidation-reduction potential some 80 mV more positive than that of normal flavin the 2-thio-FMN enzyme behaves in practically all respects like the native enzyme. It catalyzes the oxidative decarboxylation of L-lactate with almost the same efficiency as the native enzyme and with similar kinetic constants for individual steps in the catalytic pathway. It forms covalent derivatives at the flavin N(5) and C(4a) positions in facile photochemical reactions analogous to those of the native enzyme. It also forms a flavin anion radical on photoreduction with 5-deazaflavin as catalyst and, as with the native enzyme. this radical is stabilized remarkably on formation of a complex with pyruvate. The spectral properties of the neutral flavin radical form of 2-thioflavin are also reported, as determined by photochemical reduction of 2-thio-FMN flavodoxin. Like native lactate oxidase, the 2-thio-FMN enzyme also forms a flavin N(5)-sulfite adduct in an equilibrium reaction with sulfite. These results demonstrate clearly with this enzyme that the native flavin may be removed and replaced by an artificial flavin, without altering the structural integrity of the protein. [ABSTRACT FROM AUTHOR]