Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase. Role of the highly conserved Tyr-223 on substrate binding and catalysis.

We have studied D-amino-acid oxidase from Rhodotorula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis. Tyr-223, one of the very few conserved residues among D-amino-acid oxidases, has been mutated to phenylalanine and to serine. Both mutants are active catalysts in turnover with D-alanine, and they are reduced by D-alanine slightly faster than wild-type enzyme. The Tyr-223 --> Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 --> Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active-site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis

Role of Arginine 285 in the Active Site of Rhodotorula gracilis d-Amino Acid Oxidase A SITE-DIRECTED MUTAGENESIS STUDY

Abstract Arg285, one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg285 result in an increase (≈300-fold) ofK m for the d-amino acid and in a large decrease (≈500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k 2), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg285 in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg285 DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg285 in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme

PLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase : EFFECT ON SCHIZOPHRENIA SUSCEPTIBILITY

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility

Kinetic mechanisms of glycine oxidase from <i>Bacillus subtilis</i>

The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and d‐proline as substrate. The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s−1, 4.2 s−1, and 3.5 s−1, respectively, with glycine, sarcosine, and d‐proline as substrate. Glycine oxidase was converted to a two‐electron reduced form upon anaerobic reduction with the individual substrates and its reductive half‐reaction was demonstrated to be reversible. The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s−1, 170 s−1, and 26 s−1, respectively, with glycine, sarcosine, and d‐proline as substrate. The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate ≈ 3 × 104 m− 1·s−1) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex. The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate‐limiting step. Although glycine oxidase and d‐amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian d‐amino acid oxidase on neutral d‐amino acids, further supporting a close similarity between these two amine oxidases

Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum

The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s−1 and 105 s−1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s−1 for BCO and 232 s−1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Δ5-6→Δ4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s−1). With oxidized SCO and BCO the rate of isomerization is much faster (≈ 300 s−1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates ≈ 250 s−1 and 1.3 s−1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme product complexes