Pig Kidney D-amino Acid Oxidase Research Articles

Structure-Function Studies and Mechanism of Action of Snake Venom L-Amino Acid Oxidases.

Snake venom L-amino acid oxidases (SV-LAAOs) are the least studied venom enzymes. These enzymes catalyze the stereospecific oxidation of an L-amino acid to their corresponding α-keto acid with the liberation of hydrogen peroxide (H2O2) and ammonia (NH3). They display various pathological and physiological activities including induction of apoptosis, edema, platelet aggregation/inhibition, hemorrhagic, and anticoagulant activities. They also show antibacterial, antiviral and leishmanicidal activity and have been used as therapeutic agents in some disease conditions like cancer and anti-HIV drugs. Although the crystal structures of six SV-LAAOs are present in the Protein Data Bank (PDB), there is no single article that describes all of them in particular. To better understand their structural properties and correlate it with their function, the current work describes structure characterization, structure-based mechanism of catalysis, inhibition and substrate specificity of SV-LAAOs. Sequence analysis indicates a high sequence identity (>84%) among SV-LAAOs, comparatively lower sequence identity with Pig kidney D-amino acid oxidase (<50%) and very low sequence identity (<24%) with bacterial LAAOs, Fugal (L-lysine oxidase), and Zea mays Polyamine oxidase (PAAO). The three-dimensional structure of these enzymes are composed of three-domains, a FAD-binding domain, a substrate-binding domain and a helical domain. The sequence and structural analysis indicate that the amino acid residues in the loops vary in length and composition due to which the surface charge distribution also varies that may impart variable substrate specificity to these enzymes. The active site cavity volume and its average depth also vary in these enzymes. The inhibition of these enzymes by synthetic inhibitors will lead to the production of more potent antivenoms against snakebite envenomation.

Engineering the Substrate Specificity of Porcine Kidney d-Amino Acid Oxidase by Mutagenesis of the “Active-Site Lid”

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.

Catalytic and structural characteristics of carp hepatopancreas d-amino acid oxidase expressed in Escherichia coli

d-Amino acid oxidase of carp ( Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg −1 protein toward d-alanine as a substrate. It showed the highest activity toward d-alanine with a low K m of 0.23 mM and a high k cat of 190 s −1 compared to 10 s −1 of the pig kidney enzyme. Nonpolar and polar uncharged d-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 α-helices and 17 β-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney d-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp d-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities.

Improvement in thermal stability and substrate binding of pig kidney D-amino acid oxidase by chemical modification.

Chemical modification was evaluated to stabilize pig kidney D-amino acid oxidase (pkDAAO), which is required for analytical determination of D-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical modification at 30 degrees C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of activity. pkDAAO was stable at less than 55 degrees C at pH 6.0, while the conjugated enzyme was stable even at 70 degrees C. In addition, the conjugated enzyme showed decreased Km values for D-amino acids. Because of these outstanding characteristics, this new material is expected to be available for use as a liquid assay reagent.

D-Amino acid oxidase: new findings.

The most recent research on D-amino acid oxidases and D-amino acid metabolism has revealed new, intriguing properties of the flavoenzyme and enlighted novel biotechnological uses of this catalyst. Concerning the in vivo function of the enzyme, new findings on the physiological role of D-amino acid oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-amino acids in animals. A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme. At the molecular level, site-directed mutagenesis studies on the pig kidney D-amino acid oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in acid-base catalysis but rather are involved in substrate interactions. The three-dimensional structure of the enzyme was recently determined from two different sources: at 2.5-3.0 A resolution for DAAO from pig kidney and at 1.2-1.8 A resolution for R. gracilis. The active site can be clearly depicted: the striking absence of essential residues acting in acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis.

The role of a conserved histidine residue, His324, in Trigonopsis variabilis D-amino acid oxidase.

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.

Glycerol-assisted Restorative Adjustment of Flavoenzyme Conformation Perturbed by Site-directed Mutagenesis

The replacement of histidine 307 with leucine in pig kidney D-amino acid oxidase perturbs its active site conformation accompanied by dramatic losses in protein-flavin interactions and enzymatic activity. However, the negative effect of this mutation on the holoenzyme structure is essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flavin affinity. Further analysis revealed that glycerol assists in the rearrangement of the protein toward its holoenzyme-like conformation together with reduction in the solvent-accessible protein hydrophobic area as demonstrated by limited proteolysis and use of affinity and hydrophobic probes. A substantial decrease in the protein-flavin interactions was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest that the perturbation of the D-amino acid oxidase active site is due to the nonpolar nature of the mutation whose negative impact on the holoenzyme structure can be overcome by glycerol-induced strengthening of protein internal hydrophobic interactions.

Studies on the kinetic mechanism of pig kidney D-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228.

Expression conditions in Escherichia coli of wild-type, Y224F, and Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 totally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate. Reoxidation of the reduced flavin results from reaction of oxygen with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.

Immunochemical studies on Rhodotorula gracilis D-amino acid oxidase.

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified from R. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.

Characterization of a fully active N-terminal 37-kDa polypeptide obtained by limited tryptic cleavage of pig kidney D-amino acid oxidase.

In order to obtain further information on the structure of D-amino acid oxidase (EC 1.4.3.3), limited proteolysis experiments have been carried out on its apo-, holo-, and holoenzyme-benzoate forms. The enzyme is unsensitive to 10% (w/w) chymotrypsin, while incubation with 10% (w/w) trypsin, under nondenaturating conditions, produces inactivation and proteolysis patterns which are different for the three forms of enzyme analyzed. These results confirm the previously reported conformational changes which occur upon binding of coenzyme to the apoprotein, and of benzoate to holoenzyme. The stable 37.0-kDa polypeptide, obtained from the apo- and holoenzyme-benzoate complex upon cleavage of a C-terminal 2.0-kDa fragment, retains full catalytic activity with unaltered kinetic parameters, and the coenzyme binding properties of the native enzyme. These results are in agreement with the tentative localization of the FAD-binding domain in the N-terminal region of the enzyme, and with the hypothesis that the function of the C-terminal region of D-amino acid oxidase could be related to the import of the enzyme into the peroxisomes, as suggested by Gould et al. (Gould, S. J., Keller, G. A., and Subramani, S. (1988) J. Cell. Biol. 107, 897-905).

8-Azidoflavins as photoaffinity labels for flavoproteins.

8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.

Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amino acid oxidase.

Pig kidney D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3] catalyzes a rapid uptake of oxygen when high concentrations (50-100 mM) of glyoxylate and the following amines are present under usual assay conditions (pH 8.3): cysteamine, 2-aminoethanol, putrescine, D,L-1-amino-2-propanol, D,L-2-amino-1-propanol, 3-amino-1-propanol, D,L-octopamine, ethylenediamine, and L-cysteine ethyl ester. Notable physiological amines that do not support a rapid O2 reaction under the above conditions include histamine, serotonin, epinephrine, norepinephrine, spermidine, spermine, and cadaverine. A more detailed kinetic investigation of the reactions involving the first four reactive amines listed above indicated that the cysteamine reaction proceeds at a rapid rate even when cysteamine and glyoxylate are present at less than millimolar concentrations, but greater than millimolar concentrations are needed in the other amine reactions in order to observe a reasonable rate. At low concentrations and pH 7.4, the cysteamine-glyoxylate substrate (presumably thiazolidine-2-carboxylic acid) reacts an order of magnitude faster than any other known D-amino acid oxidase substrate. Considerable circumstantial evidence suggests that the reaction involving cysteamine is occurring physiologically, but the reactions of other amines would be occurring in the cell at a very low rate, if at all. It is proposed that the product of the enzymic reaction may be a metabolic effector that can modify the reactivity of proteins or nucleic acids by covalent attachment.

A simplified procedure for the isolation of pig kidney d-amino acid oxidase

A simple, quick procedure for the isolation of pig kidney d-amino acid oxidase [EC 1.4.3.3; d-amino acid:oxygen oxidoreductase (deaminating)] is described based upon the use of granulated hydroxylapatite for chromatography. The purity appears to be comparable to that obtained by other procedures. The holoenzyme was isolated as the benzoate complex.

Reactions of β-fluoroalanine and β-bromoalanine with D-amino acid oxidase

Summary D,L-β-Bromoalanine hydrobromide has been prepared from D,L-β-chloroalanine hydrochloride, and shown to be a good substrate for pig kidney D-amino acid oxidase, undergoing the oxygen independent elimination of HBr oxclusively. D-Fluoroalanine, however, undergoes solely the normal oxidation reaction to fluoropyruvate.

The Manometric Assay of d-Amino Acid Oxidase in Chicken Tissue

The original methods for measuring d-amino acid oxidase in animal tissues involved the use of either phosphate buffer (Krebs, 1935), phosphate buffer with added arsenite (Rodney and Garner, 1938), both at about pH 7.5, or pyrophosphate buffer at pH 8.6 (Krebs, 1939). The two papers found which contain values for this enzyme in chicken tissue (Trufanov and Pavlova, 1951; Williams et at., 1949) described methods employing phosphate buffer. A study of the effect of buffer and pH on the assay of purified pig kidney d-amino acid oxidase by Walaas and Walaas (1949) revealed that pyrophosphate buffer at pH 8.3 gave optimum results. Pyrophosphate buffer appears to have been used more frequently than phosphate buffer in mammalian studies (Burton, 1955). This note presents the results of comparisons of these and other buffer systems used in the measurement of this enzyme in chicken tissue.The buffer systems were compared in the following …