Biosynthesis of porcine kidney D-amino acid oxidase

The biosynthesis of a porcine kidney peroxisomal enzyme, D-amino acid oxidase (EC 1.4.3.3., DAO), was investigated. Pig kidney mRNA as well as free and membrane-bound polysomes were used to investigate in vitro protein synthesis using a rabbit reticulocyte lysate. mRNA and free polysomes, but not membrane-bound polysomes, directed the synthesis of DAO. To examine the in vivo synthesis of the enzyme, a pig kidney cell line (LLC-PK1) was biosynthetically labelled. Both the in vitro and in vivo synthesized DAO had the same molecular weight, 38,000, as that of the purified enzyme. These results indicate strongly that DAO is synthesized on free ribosomes and transferred to the interior of peroxisomes without any proteolytic modification.     

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.     

Gene expression of D-amino acid oxidase in rabbit kidney

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.     

19F-NMR study on the interaction of fluorobenzoate with porcine kidney D-amino acid oxidase

The interactions of competitive inhibitors, o-, m-, and p-fluorobenzoates, with porcine kidney D-amino acid oxidase (DAO) were studied by 19F-NMR spectroscopy. The 19F-signals of DAO-bound fluorobenzoates were observed as considerably broadened peaks. The chemical shifts, which are referenced to 20 mM NaF in 50 mM sodium phosphate, pH 7.0, were 6.0, 8.2, and 11.9 ppm for free o-, m-, and p-fluorobenzoates, respectively, while those of o-, m-, and p-fluorobenzoates bound to DAO were 12.5, 5.4, and 13.1 ppm, respectively. The 19F-signals of bound o- and p-fluorobenzoates were downfield-shifted relative to those of the free species, whereas the 19F-resonance of m-fluorobenzoate was up-field shifted from that of the free ligand. The magnitude of the chemical shift difference between the free and bound forms decreases in the order of o-, m-, and p-fluorobenzoates. The remarkably large downfield shift of the o-fluorobenzoate when bound to DAO was attributed to the close proximity of the ortho-fluorine atom to the flavin nucleus in comparison with meta- or para-fluorine. The pH-dependences of the 19F-resonances of o-, m-, and p-fluorobenzoates were observed and the pKa values of 3.33, 3.80, and 4.05 were obtained for the carboxyl groups of o-, m-, and p-fluorobenzoates, respectively. It was observed that the 19F-resonances of o- and p-fluorobenzoates are highly sensitive to the ionic state of the carboxyl group, while that of m-fluorobenzoate was moderately sensitive.     

中枢神经系统D-氨基酸氧化酶的研究进展

新近研究证实,哺乳动物神经系统中存在内源性D-氨基酸氧化酶,参与脑内D-氨基酸的代谢.遗传学研究发现,D-氨基酸氧化酶基因与精神分裂症的发生密切相关.分子生物学研究表明,在D-氨基酸氧化酶基因启动子区域存在一些转录因子的结合位点.这些研究结果提示,中枢神经系统的D-氨基酸氧化酶除了参与D-氨基酸的代谢以外,可能还具有其它的生理功能.本文就中枢神经系统D-氨基酸氧化酶的研究进展作一综述.     

Vinylglycine and proparglyglycine: complementary suicide substrates for L-amino acid oxidase and D-amino acid oxidase.

Proparglyglycine (2-amino-4-pentynoate) and vinylglycine (2-amino-3-butenoate) have been examined as substrates and possible inactivators of two flavo enzymes, D-amino acid oxidase from pig kidney and L-amino acid oxidase from Crotalus adamanteus venom. Vinylglycine is rapidly oxidized by both enzymes but only L-amino acid oxidase is inactivated under assay conditions. The loss of activity probably involves covalent modification of an active site residue rather than the flavin adenine dinucleotide coenzyme and occurs once every 20000 turnovers. We have confirmed the recent observation (Horiike, K, Hishina, Y., Miyake, Y., and Yamano, T. (1975) J, Biochem. (Tokyo), 78, 57) that D-proparglglycine is oxidized with a time-dependent loss of activity by D-amino acid oxidase and have examined some mechanistic aspects of this inactivation, The extent of residual oxidase activity, insensitive to further inactivation, is about 2%, at which point 1.7 labels/subunit have been introduced with propargly[2-14C]glycine as substrate. L-Proparglyclycine is a substrate but not an inactivator of L-amino acid oxidase and the product ahat accumulats in the nonnucleophilic N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer is acetopyruvate. In the presence of butylamine HCl, a species with lambdaman 317 nm (epsilon = 15 000) accumulates that may be a conjugated eneamine adduct. The same species accumulates from D-amino acid oxidase oxidation of D-propargylglycine prior to inactivation; the inactivated apo D-amino acid oxidase has a new peak at 317 nm that is probably a similar eneamine. A likely inactivating species is 2-keto-3,4-pentadienoate arising from facile rearrangement of the expected initial product 2-keto 4 pentynoate. Vinylglycine and proparglyglycine show inactivation specificity, then, for L-and D-amino acid oxidase, respectively.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.     

Expression of normal and abnormal porcine kidney D-amino acid oxidase in Escherichia coli: purification and characterization of the enzymes

Expression plasmids for normal and abnormal porcine D-amino acid oxidases (E.C. 1.4.3.3, DAO) have been constructed from cloned cDNA that encodes the entire protein sequence of DAO, and the enzymes were expressed in Escherichia coli cells on a large scale. The expressed enzymes were purified to apparent homogeneity. The molecular weight of the normal DAO (38 kD) was identical with that of DAO purified from porcine kidney, whereas that of the abnormal DAO was 39 kD, which comprised the normal DAO with an additional decapeptide at its amino terminus. However, the specific activities of the two enzymes were comparable with that of natural DAO. The results indicate that the bulky decapeptide does not affect the structure necessary for the catalytic function of DAO in the amino-terminal region. The use of a GTG triplet in the 5'-untranslated region of DAO cDNA as the initiation codon for the synthesis of the abnormal DAO is suggested.