Yeast D-amino acid oxidase: structural basis of its catalytic properties

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.     

The presence of high concentrations of free D-amino acids in human saliva

Free neutral D-amino acids have previously been detected in human plasma, usually accounting for less than 2% of the total free amino acid concentration (D-amino acid ratio) [Nagata, Y., Masui, R., Akino, T., 1992a. The presence of free D-serine, D-alanine and D-proline in human plasma. Experientia 48, 986-988. Nagata, Y., Yamamoto, K., Shimojo, T., 1992b. Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. Journal of Chromatography 575, 147-152. Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y., Akino, T., 1992c. The presence of free D-alanine, D-proline and D-serine in mice. Biochimca et Biiophysica Acta 1115, 208-211]. In the present study to search for the source of free D-amino acids, D- and L-enantiomers of the major non-essential amino acids, i.e., the free form of serine, alanine, proline, aspartate and glutamate were analyzed by HPLC in human saliva, submandibular glands and oral epithelial cells. The D-enantiomer ratios to total of free alanine or proline were 35% and 20%, respectively, in saliva. The ratios of the other D-amino acids were less than 11%. The effect of ingested food and oral bacteria on the saliva amino acid levels was suggested to be insignificant. D-Alanine and d-aspartate were also detected in the submandibular gland in ratios up to 5%, and D-alanine and d-proline were found in oral epithelial cells in ratios of 18% and 5%, respectively. The submandibular gland and oral epithelial cells are suggested to be possible sources of the saliva D-alanine and D-aspartate.     

Structural basis of D-DOPA oxidation by D-amino acid oxidase: alternative pathway for dopamine biosynthesis

D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO.     

The structure of a bacterial L-amino acid oxidase from Rhodococcus opacus gives new evidence for the hydride mechanism for dehydrogenation

l-Amino acid oxidase from Rhodococcus opacus (roLAAO) is classified as a member of the GR(2)-family of flavin-dependent oxidoreductases according to a highly conserved sequence motif for the cofactor binding. The monomer of the homodimeric enzyme consists of three well-defined domains: the FAD-binding domain corresponding to a general topology throughout the whole GR(2)-family; a substrate-binding domain with almost the same topology as the snake venom LAAO and a helical domain exclusively responsible for the unusual dimerisation mode of the enzyme and not found in other members of the family so far. We describe here high-resolution structures of the binary complex of protein and cofactor as well as the ternary complexes of protein, cofactor and ligands. This structures in addition to the structural knowledge of snake venom LAAO and DAAO from yeast and pig kidney permit more insight into different steps in the reaction mechanism of this class of enzymes. There is strong evidence for hydride transfer as the mechanism of dehydrogenation. This mechanism appears to be uncommon in a sense that the chemical transformation can proceed efficiently without the involvement of amino acid functional groups. Most groups present at the active site are involved in substrate recognition, binding and fixation, i.e. they direct the trajectory of the interacting orbitals. In this mode of catalysis orbital steering/interactions are the predominant factors for the chemical step(s). A mirror-symmetrical relationship between the two substrate-binding sites of d and l-amino acid oxidases is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the residues in the active site. These results are of general relevance for the mechanism of flavoproteins and lead to the proposal of a common dehydrogenation step in the mechanism for l and d-amino acid oxidases.     

Kinetic Resolution of 3-Fluoroalanine Using a Fusion Protein of D-Amino Acid Oxidase with Vitroscilla Hemoglobin

In this study, a fusion protein (VHb-DAAO) of D-amino acid oxidase (DAAO) with Vitreoscilla hemoglobin (VHb) was functionally expressed in Escherichia coli and purified. The k _cat value VHb-DAAO (47.1 s^?1) towards rac-3-flouroalanine was about 2-fold higher than that of DAAO (21.9 s^?1). rac-3-Flouroalanine (500 mM) was kinetically resolved into (R)-3-fluoroalanine with high enatiomeric excess (>99%) by VHb-DAAO with about 52% conversion.     

High-throughput screening assay for D-amino acid oxidase

A membrane-based high-throughput screening (HTS) assay for active d-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.     

Levels of D-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. d-Serine is synthesized from l-serine by the pyridoxal-5′ phosphate-dependent enzyme serine racemase (SRR), and d-serine is metabolized by d-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, d-serine, l-serine, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) mice. Levels of d-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of l-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of d-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of d-serine in the kidney, testis, and muscle of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for d-serine production in the mouse forebrain, and that other pathways of d-serine production may exist in the brain and peripheral organs.     

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

A kinetic study of D-glucose oxidation by bromine in aqueous solutions

The kinetics of the oxidation of D-glucose to D-gluconic acid by bromine in aqueous solution were studied using potentiometric techniques and theoretical considerations of complex bromine-bromide-pH equilibria. The pH has a strong influence on reaction rate. At pH < 8 the reaction is very slow, while in the pH range pH 8-9.5 the reaction is sufficiently fast and seems optimal for the reaction. The proposed active species at that pH region is hypobromous acid. At pH > 9.5, the reaction is further accelerated due to the formation of hypobromite. The proposed kinetics expression for gluconic acid formation, based on the determined kinetic parameters at pH 9.24, is of the form dc(GA)/dt = 160c(2)(G)c(o)(HOBr)c(o)(H(+)c(o)(Br)     

Enzymatic synthesis of D-glucosaminic acid from D-glucosamine

D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides and a chiral synthon, is easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) catalysed by glucose oxidase.     

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars d-allose-6-phosphate (All6P) and d-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-d-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.