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Tyr435 in human monoamine oxidase B (MAO B) was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity to probe the significance of the structure. The results provide new insights into the role of these aromatic residues in the catalytic reductive phase of amine oxidation.

Research has been conducted on the Mycobacterium tuberculosis oxidoreductase FprA. The crystal structure of FprA has been investigated in different redox states obtained via oxidized enzyme cocrystallization with NADP (super)+ and via soaking the crystals in NADPH-containing solution and the results are reported.

The ferredoxin- and NADPH-dependent glutamate synthases differ in the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S]cluster. The binding of reduced ferredoxin by the ferredoxin-dependent enzyme is required for its activities with respect to glutamate binding, hydrolysis, or ammonia transfer.

Structural analysis of the FAD-bound mutant L-aspartate oxidase reveals that FAD binding leads to a rearrangement of two polypeptide segments and a rotation of the capping domain by 27 degrees, forming active site cavity. Data indicate that the cavity surface is lined by H-bond donors anchoring the carboxylate ligands.

Site-directed mutagenesis of L-aspartate oxidase, which behaves as a fumarate reductase in anaerobic conditions, reveals that R386, H244, and H31 are involved in the fumarate binding and catalysis while R290 is involved only in the catalysis. Data show that L-aspartate active site is similar to that of fumarate reductase.

Research has been conducted on the polyamine oxidase (PAO). The crystal structure of maize PAO substrate specificity in the reduced state and and in complex with three other inhibitors has been examined to study the mechanism of amine oxidation and the PAO substrate specificity.

Research shows that a highly distorted cofactor can be bound by D-amino acid oxidase (DAAO), helping the enzyme to act as a highly efficient oxidase. DAAO also has a flexible substrate binding site. It would appear from the imino tryptophan binding mode that direct hydride transfer to the flavin N5 atom from C-alpha causes substrate oxidation. Catalysis is probably assisted by loop closure as the hydrophobicity of the active site is enhanced to improve the hydride transfer reaction. A crystallographic analysis is presented.

The turnover rate of D-amino acid oxidase (DAAO) is controlled by the release rate of the DAAO's iminopyruvate product which is determined by the movement of the active site loop to an 'open' position from a 'closed' position. Substrate specificity is also controlled by the loop. A nicked D-amino acid oxidase species is formed through the 13.4 and 25 kDa polypeptides which are produced from the limited proteolysis of D-amino acid oxidase holoenzyme with trypsin. Altered catalytic efficiency is exhibited by the nicked protein form toward D-amino acid oxidation in comparison with native DAAO.

The reduced enzymatic activity of two mutants, S558A and H610C, in Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p) is because of the absence of the hydroxyl and imidazole side chains. The activation of proton, transition state stabilization and proton transfer are performed by the A. vinelandii E2p residues Asn614', Ser558 and His610' respectively. In one class of acetyltransferase, a His-Asp-Arg triad causes optimal activity and the second class, a His-Asn pair is catalytically effective.

X-ray studies help determine crystal structures of wild-type P-hydroxy benzoate hydroxylase, complexed with substrates with amino and hydroxy groups, and of a Tyr 222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Removal of Tyr222 from the active center of Tyr 222Ala mutant does not influence the crystal structure. Substituting thr 4-hydroxy group of substrates with a 4-amino group induces binding of an extra water molecule adjacent to Tyr385, producing a hydrogen-bonding network proton channel. Binding of substrates with 2-hydroxyl group induces the displacement of flavin ring from the active site, altering its configuration.

The substrate binding and catalysis of Azotobacter vinelandii dihydrolipoyl transacetylase (E2p) were investigated. Several crystallographic binding sites were carried out by soaking the crystals with the substrates acetylCoA and dihydrolipoamide (Lip(SH)2). The results of the three-dimensional structure of the catalytic domain of E2p and the analysis of the amino acid sequences suggest that the nature and size of only a few side chains are the major determinants for tuning substrate specificity among the various transacylases.

A comprehensive structural, biochemical, and cellular study was conducted to probe the potential of enzymes LSD1 and LSD2 histone demethylases for epigenetic therapies. The data revealed potential relevance of LSD1/2 inhibitors for the treatment of promyelocytic leukemia and as tools to alter chromatin state with promise of a block of tumor progression.

The structural properties and mechanisms of the C-terminal transmembrane helices membrane-bound mitochondrial monoamine oxidases MAO A and MAO B, which are pharmacological targets for specific inhibitors, are reported. The structural and mechanistic information related to highly specific inhibitorsof MAO A and MAO B could find potential use for the future development of highly specific neuroprotectants and cardioprotectants.

The results from comparative analysis of the structure and mechanism of human monoamine oxidase B (MAO B) inhibition by hydrazines with data on human monoamine oxidase A (MAO A) is presented. Both enzymes were found to be inhibited by similar [O.sub.2]-mediated mechanisms.

The crystal structure analysis of the recombinant enzyme, alditol oxidase (AldO), isolated from Streptomyces coelicolor is reported.

The properties of mutant Mycobacterium tuberculosis FprA, a NADPH-ferredoxin reductase, and the role of the His-57 and Glu-214 ionic couple located in the active site of M. tuberculosis are described. The results show that His-57 is important in the correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high rate.

DFT calculations presented for C(4a)-hydroperoxyflavin (C(4a)-FLHOOH) at the B3LYP/6-311+G(d,p) level suggest a new mechanism for the elimination of H2O2. The calculated activation barrier for a concerted four-centered elimination (ΔE‡ = 32.86 kcal/mol) strongly suggests that in the absence of interactions with the local environment a spontaneous elimination is not feasible. A proton shuttle from the N5 hydrogen to the proximal oxygen of the OOH moiety involving three water molecules has an activation barrier that is reduced to 17.11 kcal/mol. Calculations that utilize CH3OH to model the role of a local Thr or Ser residue shows that an alcohol functionality hydrogen bonded to the N5 H-atom can catalyze the elimination of H2O2 with a free energy of activation of 21.5 kcal/mol. Interaction of amines and amide residues (CH3NH2 and CH3(C=O)NH2) with the N5 locus of C(4a)-hydroperoxyflavin markedly reduce the activation barrier for H2O2 elimination relative to the concerted pathway. Proton transfer from a COOH group (ΔG‡ = 8.36 kcal/mol) or the NH2 group of a positively charged Arg model (ΔG‡ = 9.99 kcal/mol) to the proximal oxygen of the OOH moiety of C(4a)-FLHOOH in the TS for H2O2 elimination strongly enhances elimination of H2O2. [ABSTRACT FROM AUTHOR]

Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 ( Hsp UPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris . The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. Hsp UPO is a versatile catalyst performing various reactions, such as the oxidation of prim - and sec -alcohols, epoxidations and hydroxylations. Semi-preparative biotransformations were demonstrated for the non-enantioselective oxidation of racemic 1-phenylethanol rac -1b (TON = 13000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. Hsp UPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with small-to-medium size substrates of variable polarities. The structural investigation of several protein-ligand complexes revealed that the active site of Hsp UPO is accessible to molecules of varying bulkiness and polarity with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, Hsp UPO is an exploitable candidate for peroxygenase-based biocatalysis., Competing Interests: Conflict of interest: The authors declare no conflicts of interest in regards to this manuscript.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O 2 in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a F 420 H 2 :NADP + -like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome b subunits of the mitochondrial complex III and photosynthetic cytochrome b 6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes b act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome b domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome b enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from Thermocrispum municipale , a flavin-dependent Baeyer-Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates., Competing Interests: The authors declare no competing financial interest.

Flavin-containing monooxygenases (FMOs) are emerging as effective players in oxidative drug metabolism. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating molecules containing soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer-Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metabolism of active pharmaceutical ingredients, we selected and tested drug molecules bearing a carbonyl group on an aliphatic chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacologically active compound. Using the recombinant enzyme, the ester derivatives of both drugs were obtained in milligram amounts, purified, and fully characterized. This protocol can potentially be extended to other FMO5 candidate substrates as it represents an effective and robust bench-ready platform applicable to API screening and metabolite synthesis.

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy.

The pure four diastereomers (11a-d) of trans-benzyl (1-((4-(2-aminocyclopropyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate hydrochloride 11, previously described by us as LSD1 inhibitor, were obtained by enantiospecific synthesis/chiral HPLC separation method. Tested in LSD1 and MAO assays, 11b (S,1S,2R) and 11d (R,1S,2R) were the most potent isomers against LSD1 and were less active against MAO-A and practically inactive against MAO-B. In cells, all the four diastereomers induced Gfi-1b and ITGAM gene expression in NB4 cells, accordingly with their LSD1 inhibition, and 11b and 11d inhibited the colony forming potential in murine promyelocytic blasts.

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or "pan-KDM" inhibitors 1-6 by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action.

DFT calculations presented for C(4a)-hydroperoxyflavin (C(4a)-FLHOOH) at the B3LYP/6-311+G(d,p) level suggest a new mechanism for the elimination of H2O2. The calculated activation barrier for a concerted four-centered elimination (ΔE(‡) = 32.86 kcal/mol) strongly suggests that in the absence of interactions with the local environment a spontaneous elimination is not feasible. A proton shuttle from the N5 hydrogen to the proximal oxygen of the OOH moiety involving three water molecules has an activation barrier that is reduced to 17.11 kcal/mol. Calculations that utilize CH3OH to model the role of a local Thr or Ser residue shows that an alcohol functionality hydrogen bonded to the N5 H-atom can catalyze the elimination of H2O2 with a free energy of activation of 21.5 kcal/mol. Interaction of amines and amide residues (CH3NH2 and CH3(C═O)NH2) with the N5 locus of C(4a)-hydroperoxyflavin markedly reduce the activation barrier for H2O2 elimination relative to the concerted pathway. Proton transfer from a COOH group (ΔG(‡) = 8.36 kcal/mol) or the NH2 group of a positively charged Arg model (ΔG(‡) = 9.99 kcal/mol) to the proximal oxygen of the OOH moiety of C(4a)-FLHOOH in the TS for H2O2 elimination strongly enhances elimination of H2O2.