Cloning and Expression in Escherichia coli of the D-Aspartate Oxidase Gene from the Yeast Cryptococcus humicola and Characterization of the Recombinant Enzyme

Takahashi, Shouji; Takahashi, Toshiyuki; Kera, Yoshio; Matsunaga, Ryuji; Shibuya, Hiroo; Yamada, Ryohei

doi:10.1093/jb/mvh068

Cited by 25 publications

(21 citation statements)

References 26 publications

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“…hDASPO shows a 5-and >10-fold higher specific activity than the homologous bovine enzyme and the orthologous flavoenzyme human d-amino acid oxidase (active on d-Ser), respectively. The oligomerization state of hDASPO differs from that of pig and yeast DASPOs (which are tetramers) 43 and from that of the homologous DAAOs and eukaryotic L-amino acid oxidases, which are stable homodimers in solution. 42 .…”

Section: Biochemical Properties Of Hdaspomentioning

confidence: 83%

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

et al. 2019

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Savinelli equally contributed to this study.Abbreviations: 5-An, 5-aminonicotinic acid; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate; CBIO, 6-chloro-1,2-benzisoxazol-3(2H)-one; DAAO, d-amino acid oxidase; DASPO or DDO, d-aspartate oxidase; DPPD, pyrido[2,3-b]pyrazine-2,3(1H,4H)-dione; EMTN, enzyme monitored turnover; hDAAO, human d-amino acid oxidase; hDASPO, human d-aspartate oxidase; IAsp, iminoaspartate; MT, meso-tartrate; NMDAR, N-methyl-d-aspartate receptor; OA, oxaloacetate; RgDAAO, d-amino acid oxidase from Rhodotorula gracilis. Abstract d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-daspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first threedimensional structure of a DASPO enzyme (from human) which belongs to the damino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR)showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target. K E Y W O R D Sd-aspartate, d-aspartate oxidase, flavoprotein, NMDA receptor, structure-function relationships

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Section: Biochemical Properties Of Hdaspomentioning

confidence: 83%

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

et al. 2019

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“…Examination of the reported kinetic properties of recombinant mammalian and micro‐organism DASPOs suggests that C47Ap and F18Ep are unusual with regard to their specificity for d ‐Glu. The catalytic efficiency of C47Ap against d ‐Glu is 23 066 s −1 · m −1 , which is 108 and 923 times higher than the respective catalytic efficiencies of bovine and Cryptococcus humicola DASPOs against d ‐Glu (213 and 25.0 s −1 · m −1 , respectively) [43,69]. Similarly, the catalytic efficiency of F18Ep against d ‐Glu is 8427 s −1 · m −1 , which is 39.6 and 337 times higher than the catalytic efficiencies of bovine and C. humicola DASPOs, respectively.…”

Section: Discussionmentioning

confidence: 99%

Caenorhabditis elegans has two genes encoding functional d‐aspartate oxidases

Katane¹,

Seida²,

Sekine

et al. 2006

The FEBS Journal

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As all amino acids except Gly have an asymmetric a-carbon atom to which both amino and carboxyl groups attach, they can exist as two kinds of stereoisomers, namely, the l-forms and d-forms. It has long been believed that d-amino acids do not play a significant role in the physiology of most organisms, apart from bacteria, where they are essential as components of cell wall peptidoglycans and antibiotic peptides. However, advances in the methods used to separate chiral amino acids have revealed that various living organisms contain several d-amino acids, either in their free form or as protein components. Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d-amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d-Arg more efficiently than d-Ala and dMet, and that C47Ap and F18Ep show distinct kinetic properties against d-Asp, d-Glu, and N-methyl-d-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.Abbreviations C47Ap, the C47A10.5 gene product; DAAO, D-amino acid oxidase; DASPO, D-aspartate oxidase; F18Ep, the F18E3.7a gene product; F20Hp, the F20H11.5 gene product; NMDA, N-methyl-D-aspartate; PTS1, type 1 peroxisomal targeting signal; PTS2, type 2 peroxisomal targeting signal; Y69Ap, the Y69A2AR.5 gene product.

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“…A molar extinction coefficient for oxidized o-dianisidine of 8.3 ϫ 10 3 M Ϫ1 cm Ϫ1 was used for calculating the enzyme activity. The DNPH assay was performed with D-valine as a substrate in 50 mM potassium phosphate buffer (pH 8.0) at 50°C, as described previously (20). The enzyme activity was determined colorimetrically at 440 nm using a molar extinction coefficient for the ketovaline hydrazone of 1.58 ϫ 10 4 M Ϫ1 cm Ϫ1 (21).…”

Section: Methodsmentioning

confidence: 99%

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...

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Cloning and Expression in Escherichia coli of the D-Aspartate Oxidase Gene from the Yeast Cryptococcus humicola and Characterization of the Recombinant Enzyme

Cited by 25 publications

References 26 publications

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Structure and kinetic properties of human d‐aspartate oxidase, the enzyme‐controlling d‐aspartate levels in brain

Caenorhabditis elegans has two genes encoding functional d‐aspartate oxidases

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

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