Stereochemistry and accessibility of prosthetic groups in flavoproteins

Using 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of the appropriate flavin nucleotides, we determined the stereochemistry of interaction between coenzyme and substrate for several flavoproteins. The enzymes were D-amino acid oxidase, L-lactate oxidase, and D-lactate dehydrogenase, all three of which interact with pyruvate, as well as cyclohexanone monooxygenase and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase, which were both probed with nicotinamide nucleotides. L-Lactate oxidase and D-lactate dehydrogenase used the si face of the modified flavin ring while the other three enzymes showed re-side specificity. This selection of flavoenzymes includes FAD- and FMN-dependent enzymes, enzymes that follow a carbanion mechanism, and others that have hydride transfer as an integral part of their reaction pathway.     

Enzymatic method for measuring the absolute value of oxygen concentration

An enzymatic method for measuring the absolute concentration of oxygen in aqueous solutions, using 4-hydroxybenzoate 3-monooxygenase and glucose oxidase, is described. The monooxygenase is used for quantitative oxidation of 4-hydroxybenzoate and NADPH with oxygen into 3,4-dihydroxybenzoate and NADP+; the amount of oxygen can be measured as the amount of NADPH decreased by the reaction. The monooxygenase reaction is performed in a syringe, a closed system. After the completion of the monooxygenase reaction, glucose oxidase is added to the assay solution to consume the oxygen from the atmosphere; this enables us to measure the NADPH concentration in the solution spectrophotometrically in an open system and to check the anaerobicity of closed systems. The oxygen concentrations at 25 degrees C of oxygen-saturated and air-saturated water were 1.10 and 0.23 mM, respectively. The value for argon-bubbled water was zero within the experimental error; this justifies the assay system. Thus, it is shown that a sample containing 8 microM-1.1 mM oxygen can be measured by this method.     

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.     

Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity

An understanding of the mechanism of biological methane oxidation has been hampered by the lack of purified proteins. We describe here a purification protocol for the previously uncharacterized protein B of the soluble methane monooxygenase from the obligate methanotroph Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme consisting of a hydroxylase component, protein A, a reductase component, protein C, and protein B. All three proteins are required for monooxygenase activity. Protein B proves to be a low molecular weight (16,000) single subunit protein devoid of prosthetic groups. The protein is a powerful regulator of soluble methane monooxygenase activity, possessing the capacity to convert the enzyme from an oxidase to an oxygenase. Proteins A and C together catalyze the reduction of molecular oxygen to water, a reaction prevented by protein B. The uncoupling of soluble methane monooxygenase in this manner displays a number of novel features. First, the product of the uncoupled reaction is water, and second, the uncoupling is independent of substrate. Free hydrogen peroxide is not an intermediate in the reduction of oxygen by the incomplete methane monooxygenase enzyme complex. Finally, electron transfer can occur between protein C and protein A in the absence of protein B and protein B prevents the steady-state transfer of electrons in the absence of an oxidizable substrate, such as methane. It is demonstrated that oxygen reduction occurs at the active site of the hydroxylase component, protein A. A unifying mechanism, describing the interaction of the three proteins of soluble methane monooxygenase, is proposed.     

Exploring the molecular nature of alternative oxidase regulation and catalysis

Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein-derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.     

Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate

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 polar 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 Lys265 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 ligands 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 of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.     

Generating disulfides with the Quiescin-sulfhydryl oxidases

The Quiescin-sulfhydryl oxidase (QSOX) family of flavoenzymes catalyzes the direct and facile insertion of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. This review discusses the chemical mechanism of these enzymes and the involvement of thioredoxin and flavin-binding domains in catalysis. The variability of CxxC motifs in the QSOX family is highlighted and attention is drawn to the steric factors that may promote efficient thiol/disulfide exchange during oxidative protein folding. The varied cellular location of these multi-domain sulfhydryl oxidases is reviewed and potential intracellular and extracellular roles are summarized. Finally, this review identifies important unresolved questions concerning this ancient family of sulfhydryl oxidases.     

Structural analysis of the catalytic mechanism and stereoselectivity in Streptomyces coelicolor alditol oxidase

Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.     

Sulfoxide reductase activity of liver aldehyde oxidase

The present study provides evidence that guinea pig and rabbit liver aldehyde oxidase (EC 1.2.3.1) in the presence of its electron donors such as aldehydes or N-heterocyclic compounds functions as a sulfoxide reductase towards sulindac and other sulfoxide compounds. In addition, the study shows that a combination of liver aldehyde oxidase and milk xanthine oxidase also exhibits sulfoxide reductase activity in the presence of xanthine, and electron donor of xanthine oxidase. Based on these facts, we propose a new electron-transfer system consisting of these two flavoenzymes.     

Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP⁺ analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this “double-engineering” approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP⁺ in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.     

GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities.

Sequence comparison of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase and Hansenula polymorpha methanol oxidase indicates that these four diverse flavoproteins are homologous, defining a new family of proteins named the GMC oxidoreductases. These enzymes contain a canonical ADP-binding beta alpha beta-fold close to their amino termini as found in other flavoenzymes. This domain is encoded by a single exon of the D. melanogaster glucose dehydrogenase gene.     

Dioxygenase- and monooxygenase-catalysed synthesis of <Emphasis Type="Italic">cis</Emphasis>-dihydrodiols,catechols, epoxides and other oxygenated products

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.     

On the reaction of D-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity

Evidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability. However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O(2) with reduced flavin, together with a fivefold decrease in the K(m) for dioxygen. This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alteration in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen. The increase in O(2) reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applications.     

Substrate binding modulates the activity of Mycobacterium smegmatis G, a flavin-dependent monooxygenase involved in the biosynthesis of hydroxamate-containing siderophores

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 k(cat) value for formation of hydroxylated l-lysine under steady-state conditions was 5.0 min(-1), and K(m) 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 k(cat) 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.     

Structural insights into the mechanism of amine oxidation by monoamine oxidases A and B

Due to their pharmacological importance in the oxidation of amine neurotransmitters, the membrane-bound flavoenzymes monoamine oxidase A and monoamine oxidase B have attracted numerous investigations and, as a result, two different mechanisms; the single electron transfer and the polar nucleophilic mechanisms, have been proposed to describe their catalytic mechanisms. This review compiles the recently available structural data on both enzymes with available mechanistic data as well as current NMR data on flavin systems to provide an integration of the approaches. These conclusions support the proposal that a polar nucleophilic mechanism for amine oxidation is the most consistent mechanistic scheme as compared with the single electron transfer mechanism.     

Correlation of kinetic parameters of nitroreductase enzymes with redox properties of nitroaromatic compounds

The well-established mechanism of regeneration of the parent nitro compound by the reaction of the nitro anion radical with oxygen in aerobic systems is the basis of the correlation of kinetic parameters of purified flavoenzymes with electron affinities of some selected nitroaryl and nitroheterocyclic compounds. We have found that there is a linear relationship between log Vmax/Km and the one-electron reduction potentials of these compounds and that the measured values of redox dependence for these compounds is similar to that determined by other methods. Our results support the proposal of a rate-determining single electron-transfer as the initial step in the reduction of nitro compounds by purified flavoenzymes and are discussed in terms of the Marcus electron transfer theory.     

An aromatic hydroxylation reaction catalyzed by a two-component FMN-dependent Monooxygenase. The ActVA-ActVB system from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.     

Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by alpha-lipoamide dehydrogenase flavin (Em7.4 = -283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme (Em7.4 = -52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, alpha-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.     

The primary structure of the flavoprotein D-aspartate oxidase from beef kidney.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.     

Theoretical approaches to D-amino acid oxidase

Several substrates and roles have been proposed for D-amino acid oxidase (E.C. 1.4.3.3.); however, there is no proof that they possess the required characteristics to account for the ubiquity, large amounts and great activity of the enzyme as found in diverse cells and tissues. Based on the similar stereoposition of identically charged atoms and lateral side chain (R) with respect to the alpha-hydrogen atoms in beta-sheet conformation and in D-amino acids, it is proposed that its substrates may include several membrane-related proteins, partially in beta-sheet conformation, whose alpha-hydrogen atoms would be the real object of D-amino acid oxidase catalysis. A monooxygenase-like enzymatic activity of D-amino acid oxidase with these novel substrates is considered, for which the final products are hypothesized to be protein alpha-carbon hydroxyls resulting from the incorporation of one atom of oxygen into the substrate, the other being reduced to water. Alternatively, it is also proposed that D-amino acid oxidase (and possibly other monooxygenase enzymes) would have a hydroperoxide-synthetase activity. In this case, protein alpha-carbon hydroperoxide and not water, but another reduced molecule, would be the final products. The new enzymatic performances of D-amino acid oxidase and the possible role of its potential final products in redox and other biochemical processes are discussed.