An active site-tyrosine-containing heptapeptide from D-amino acid oxidase

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.     

An enzyme electrode for amperometric measurement of D-amino acid

A carbon paste enzyme electrode has been developed for measurement of D-amino acids that employs a fatty acid modified FAD to prevent leaching of this essential cofactor to the surrounding aqueous environment and which serves as an enzyme stabilizing agent. The lower limit of detection is at least 10(-4) M and the electrode has a linear range of 10(-4) to 3 x 10(-3) M and a response time of 180 s. Twenty L-amino acids were tested and none of them elicited responses when electrodes were exposed to 0.5 mM concentration increases over a baseline level. On the other hand, some response was observed when exposed to 18 of 20 D-amino acids varying from 2 to 200% of the corresponding D-alanine response. Electrodes were shown to have longevities of over 30 days while maintaining 85% of their original sensitivity. Electrodes showed activity over a pH of 6.2-11.7 with a maximum at 9.2 and over temperatures of 10-47 degrees C with a maximum at 37 degrees C.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

D-amino acid oxidase in mouse liver--II

1. Activity of D-amino acid oxidase was detected in tissue extract of mouse liver by two sensitive spectrophotometric methods. 2. The activity was also detectable in extracts of the heart, but not of lung.     

Second lytic target of beta-lactam compounds that have a terminal D-amino acid residue

A new biochemical mechanism of lysing bacterial cells by treatment with certain beta-lactam compounds that possess a terminal D-amino acid moiety in their side chain was demonstrated. The two functions of the molecule, the beta-lactam and terminal D-amino acid moiety, are both involved in the activity of lysing gram-negative bacteria, which is characterized by very rapid lysis of the cells in the first few hours after their contact with the compound. This mechanism was proved by studies on one such compound, named MT-141, which contains a terminal D-cysteine moiety with free amino and carboxyl groups in the 7 beta-side chain of the 7 alpha-methoxy-cephalosporin skeleton. This compound bound to the cell-wall peptidoglycan of Escherichia coli through the D-amino group of its terminal D-amino acid moiety and this seemed to cause rapid cell lysis. Both activities, of binding to peptidoglycan and of causing rapid cell lysis, were inhibited by certain D-amino acids, but not by L-amino acids. Mutants were isolated that had simultaneously gained decreased sensitivity to this kind of beta-lactam compound and supersensitivity to globomycin, an inhibitor of formation of lipoproteins which function in linking the peptidoglycan to the outer membrane. These results suggest that binding of the terminal D-amino acid moiety of the beta-lactam compound to peptidoglycan somehow influences formation of the linkage between the outer membrane and the peptidoglycan and consequently enhances the cell lytic activity of the beta-lactam portion of the molecule.     

D-amino acid oxidase in mouse liver

1. An appreciable amount of D-amino acid oxidase was found in the extract of mouse liver by enzyme-linked immunosorbent assay (ELISA). 2. The content of the enzyme in the kidney and heart extracts was also measured by the assay.     

Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Mutations distant from the tryptophanase gene.

Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.     

D-amino acid residues in peptides and proteins

We have investigated the D-amino acid residues present in Protein Data Bank (PDB) entries, categorizing them into "real" D-residues and artifacts. In polypeptide chains of more than 20 residues, only a single instance of a "real" D-residue, other than those deliberately designed or engineered, was found. This example was the result of a slow chemical epimerization process. Another 12 designed D-residues were found in these longer polypeptide chains. Smaller peptides of 20 or fewer residues contained 479 "real" D-residues, the majority in various gramicidin, actinomycin, or cyclosporin structures. We found 148 PDB entries with "real" D-residues and a further 186, in which all apparent D-residues are artifacts. Investigating the (phi, psi) preferences of the "real" D-residues, we found that the region around (-60 degrees, -45 degrees ) was almost completely unoccupied, even though it is not formally disallowed. We link the low propensity to occupy this region with the alpha-helix destabilizing properties of D-residues.     

Involvement of D-amino acid oxidase in elimination of free D-amino acids in mice.

The physiological role of D-amino acid oxidase was investigated by using mutant ddY/DAO- mice lacking the enzyme. Free D-amino acid concentrations in the mutant mice were significantly higher than those of control ddY/DAO+ mice in kidney, liver, lung, heart, brain, erythrocytes, serum and urine. The results suggest that the enzyme is involved in the catabolism of free D-amino acids in the body, and that free D-amino acids are also excreted into urine.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

Reactivity of D-amino acid oxidase with 1,2-cyclohexanedione: evidence for one arginine in the substrate-binding site

D-Amino acid oxidase is inactivated by reaction with 1,2-cyclohexanedione in borate buffer at pH 8.8. The reaction follows pseudo-first-order kinetics. The present of benzoate, a substrate-competitive inhibitor of the enzyme, protects substantially against inactivation. Partial reactivation could be obtained by removal of borate and its substitution with phosphate buffer. The reaction of 1,2-cyclohexanedione with the enzyme at different inhibitor concentrations appears to follow a saturation kinetics, indicating the formation of an intermediate complex between enzyme and inhibitor prior to the inactivation process. The partially inactivated enzyme shows the same apparent Km but a decreased V as compared to the native D-amino acid oxidase. Similarly, the inhibited enzyme fails to bind benzoate. Amino acid analysis of the 1,2-cyclohexanedione-treated enzyme at various times of inactivation shows no loss of amino acid residues except for arginines. Analysis of the reaction data by statistical methods indicates that three arginine residues react with the inhibitor at slightly different rates, and that one of them is essential for catalytic activity. The presence of benzoate, while it prevents the loss of activity, reduces by one the number of arginine residues hit by the reagent in the reaction of 1,2-cyclohexanedione with D-amino acid oxidase.     

Occurrence of D-amino acid aminotransferase in pea seedlings

Aminotransferase reacting between D-amino acids and α-keto acids was found in the germinating pea seedlings. With partial purification, it was evident that the enzyme preparation contained no racemase and L-amino acid aminotransferase and was only specific for D-amino acid transamination. Large amounts of D-alanine found in the germinating pea seedlings were assumed to be formed by this enzyme action.