Flavoprotein Oxidases Research Articles

Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila.

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

121 - Redox Control of the Critical Immune Regulatory Enzyme Indoleamine 2,3-Dioxygenase by NADPH Oxidase 2-Derived Reactive Oxygen Species in Human Monocytes

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a critical immune regulatory enzyme capable of signalling for immune suppression in diverse pathological settings including chronic inflammation, infection and cancer. During inflammation, IDO1 is expressed within activated innate immune cells that also produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) by NADPH oxidase (Nox) and myeloperoxidase (MPO), respectively. We recently reported that H2O2 inhibits recombinant IDO1 dioxygenase activity by engaging the enzyme’s heme peroxidase activity resulting in oxidative self-inactivation (JBC 2013;288:1548). To test if Nox-derived H2O2 or MPO-derived HOCl also control cellular IDO1 activity, we studied IFN-γ-stimulated human monocytes that co-express IDO1, Nox and MPO. Stimulation of monocytes with PMA increased cellular production of H2O2 and HOCl that coincided with the post-translational inhibition of IDO1 activity. The ability of PMA to inhibit IDO1 was abrogated by inhibition of Nox-derived H2O2 production with the PKC inhibitor Bisindolylmaleimide or the flavoprotein oxidase inhibitor diphenyleniodonium, but not by inhibition of MPO-derived HOCl with 4-aminobenzoic acid hydrazide. IFN-γ-stimulated human monocytes not only expressed the phagocytic Nox2 isoform but also Nox4. While selective Nox4 inhibition reduced cellular H2O2 production by ~50%, it did not reverse PMA’s inhibitory action on IDO1. Also, PMA did not inhibit IDO1 activity in IFN-γ-stimulated monocytes isolated from chronic granulomatous disease (CGD) patients expressing defective Nox2. These studies establish that Nox2-derived ROS control IDO1 activity in human innate immune cells and provide important new insights into the redox control of IDO1’s immune regulatory actions.

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.

Biosynthesis of Shearinine: Diversification of a Tandem Prenyl Moiety of Fungal Indole Diterpenes.

The late-stage biosynthetic pathway of the indole diterpene shearinine involving four enzymatic reactions (JanQDOJ) was elucidated by an efficient heterologous expression system using Aspergillus oryzae. Key oxidative cyclization, forming a characteristic A/B bicyclic shearinine core by flavoprotein oxidase, was studied using a substrate analogue and a buffer containing H218O. These experimental data provided evidence that JanO catalyzes two-step oxidation via a hydroxylated product and that the JanO reaction involves the hydride-transfer mechanism.

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst.

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.

Evidence for catalytic intermediates involved in generating the chromopyrrolic acid scaffold of rebeccamycin by RebO and RebD

We provide the first experimental evidence for intermediates being involved in catalysis by RebD in generating the chromopyrrolic acid (CPA) scaffold of rebeccamycin. In the presence of its substrates (indole pyruvate imine – IPAI – and H2O2 both produced by the flavoprotein oxidase RebO that oxidizes tryptophan), RebD reacts as a peroxidase forming two IPAI radicals that recombine as a C–C bond in the CPA. When catalase is included to remove H2O2, CPA can still be formed because the IPAI rapidly reduces RebD, which reacts with O2, utilizing oxidase–peroxidase chemistry to produce CPA. Reduced RebD can also react with H2O2 forming Cpd II directly, which can oxidize IPAI. Stopped-flow spectrophotometric studies demonstrated that during the reaction of RebO and RebD with Trp and oxygen, a species with a red-shifted Soret band at 424.5nm appeared. This species can react with either guaiacol or ABTS to form ferric RebD, suggesting that it is Cpd II of RebD involved in the formation of CPA. In summary, the studies reveal new and unusual aspects peroxidase and peroxygenase chemistry used by RebD in catalyzing carbon–carbon oxidative coupling reactions that are involved in biosynthesis of indolocarbazoles.

Amine Oxidation Mediated by N-Methyltryptophan Oxidase: Computational Insights into the Mechanism, Role of Active-Site Residues, and Covalent Flavin Binding

Amine oxidation, a process widely utilized by flavoprotein oxidases, is the rate-determining step in the three-step demethylation of N-methyltryptophan (NMT) catalyzed by N-methyltryptophan oxidase (MTOX), which employs a covalently bound flavin adenine dinucleotide (FAD) as cofactor. For the required transfer of a hydride ion equivalent, three pathways (direct/concerted, radical, and adduct-forming/polar nucleophilic) have been proposed, without a consensus on which one is commonly used by amine oxidases. We combine theoretical pKa analysis, classical molecular dynamics (MD) simulations, and pure quantum mechanics (QM) and hybrid QM/molecular mechanics (QM/MM) calculations to provide molecular-level insights into the catalytic mechanism of NMT oxidation and to analyze the role of MTOX active-site residues and covalent FAD incorporation for NMT binding and oxidation. The QM(B3LYP-D2/6-31G(d))/CHARMM results clearly favor a direct concerted hydride transfer (HT) mechanism involving anionic NMT as the reactive species. On the basis of classical canonical MD simulations and QM/MM calculations of wild-type MTOX and two mutants (K341Q and H263N), we propose that the K341 residue acts as an active-site base and electrostatically, whereas H263 and Tyr249 only support substrate alignment. Covalent FAD binding leads to a more bent isoalloxazine moiety, which facilitates the binding of anionic NMT but increases the catalytic activity of FAD only slightly.

The oxidation of thiols by flavoprotein oxidases: a biocatalytic route to reactive thiocarbonyls.

Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of C-O, C-N, or C-C bonds. Flavoprotein oxidases that are known to catalyze the oxidation of C-S bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.

The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls

AbstractFlavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of CO, CN, or CC bonds. Flavoprotein oxidases that are known to catalyze the oxidation of CS bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.

83: S100A8 is induced in macrophages by oxidants and modulates redox responses in inflammation

S100A8 expression is modulated by stressors that promote reactive oxygen species (ROS) generation. Its induction in macrophages activated by TLR-4, −7, and −9 ligands is dependent on IL-10 production. S100A8 participates in the inflammatory response at various levels, including scavenging of oxidants and nitric oxide. Inflammation increases ROS that can mediate cell signaling to control gene expression. Macrophages treated with LPS in vitro produce ROS and induces S100A8. IL-10 and S100A8 induction were both inhibited by the antioxidant N-acetyl-cysteine, the flavoprotein oxidase inhibitor diphenylene iodonium and the NADPH oxidase inhibitor apocynin. The actions of these pharmacological agents on S100A8 gene expression were recapitulated by downregulation of expression of NADPH oxidase 2 using siRNA, and studies with peritoneal macrophages from NADPH oxidase 2 −/− mice. Importantly, although H 2 O 2 and IL-10 did not induce IL-10 or S100A8 individually when combined they promoted strong expression of both genes. We next examined whether S100A8 modulated genes involved in ROS generation in LPS-mediated lung inflammation. Q-PCR arrays containing 30 redox-related genes, showed that intranasal inhalation of S100A8 directly induced itself and IL-10 in airway epithelial cells, and heme oxygenase-1 and arginase 2 mRNAs. In mice with LPS-induced lung injury pretreated with S100A8 inhalation, mRNA levels of antioxidant defense proteins, including catalase, metallothioneine-1, glutathione peroxidase, glutathione S-transferase and thioredoxin in lung tissue were potentiated, and iNOS was reduced, compared to lungs from mice treated with LPS alone. Modulation by S100A8 was at least partially dependent on its reactive thiol residue. Cys 42 to Ala 42 mutant of S100A8 did not induce IL-10 in the lung, and effects on antioxidant genes were reduced. We propose a regulatory feedback loop whereby IL-10 and S100A8 levels are influenced by ROS generation to modulate the redox network in resolution of inflammation. Support: Funded by Grants #630647 and #1027189 , NHMRC Australia .

Convenient microtiter plate‐based, oxygen‐independent activity assays for flavin‐dependent oxidoreductases based on different redox dyes

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs.

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX) is the major Ca(2+) efflux pathway on the sarcolemma, counterbalancing Ca(2+) influx via L-type Ca(2+) current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca(2+) load and can contribute to abnormal Ca(2+) handling and arrhythmias. NADH/NAD(+) is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD(+) redox potential regulates NCX activity in adult cardiomyocytes. NCX current (INCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced INCX inhibition was abolished by the H2O2 scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because INCX was insensitive to cytosolic NADPH, and NADH-induced ROS and INCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD(+) redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca(2+) homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.

Flavoprotein oxidases: classification and applications

This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.

Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 μm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Applications of Flavoprotein Oxidases in Organic Synthesis: Novel Reactivities that Go Beyond Amine and Alcohol Oxidations

Oxidases represent a distinct and interesting class of oxidative biocatalysts. A major portion of the known oxidases contain a flavin as cofactor, with glucose oxidase as best known example. While a number of oxidases are well known in the field of biocatalysis, the total number of available oxidases is rather limited. However, by analysis of literature data and genome sequences a clear picture emerges: nature harbors a large number of unexplored flavoprotein oxidases that can catalyze a breadth of oxidative reactions. In this review a summary is provided of the unique and intriguing catalytic potential of newly discovered flavoprotein oxidases. The reactions that have been shown to be catalyzed range from enantio- and/or regioselective alcohol and amine oxidations to oxidative C-O or C-C bond formations. This illustrates that nature harbors a large collection of oxidases that can be of use for catalyzing complex oxidative reactions. Keywords: Alcohol, Biocatalysis, C-C bond, Cyclization, Oxidases, Flavoprotein

A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii

D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30°C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33°C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38°C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30°C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. Km and Vmax values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform.