Porcine kidney D-amino acid oxidase

Chemical synthesis and isolation of 2-keto-6-hydroxyhexanoic acid required several steps. In a second, more convenient process (Fig. 2), the ketoacid was prepared by treatment of racemic 6-hydroxynorleucine [produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin (3)] with D-amino acid oxidase and catalase. After the e.e. of the remaining L-6-hydroxynorleucine had risen to >99%, the reductive animation procedure was used to convert the mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine entirely to L-6-hy-droxynorleucine with yields of 91-97% and e.e. of >98%. Sigma porcine kidney D-amino acid oxidase and beef liver catalase or Trigonopsis variabilis whole cells (source of oxidase and catalase) were used successfully for this transformation [22],... 

Mizutani, H., Miyahara, I., Hirotsu, K., Nishima, Y., Shiga, K., Setoyama, C., and Miura R., 1996, Three-dimensional structure of porcine kidney D-amino acid oxidase at 3 resolution, J. Biochem. 120 14nl7. 

L-6-hydroxynorleucine with yields of 91 to 97% and ee > 99%. Sigma porcine kidney D-amino acid oxidase and beef liver catalase or Trigonopsis variabilis whole cells (source of oxidase and catalase) were used successfully for this transformation. 

Fukui K, Watanabe F, Shibata T, Miyake Y. Molecular cloning and sequence analysis of cDNA encoding porcine kidney D-amino acid oxidase. Biochem 1987 26 3612-3618. 

Porcine kidney D-amino acid oxidase (pkDAAOx) was identified as the first mammalian flavoprotein to catalyze the oxidative deamination of a-amino acids with strict i -stereoselectivity. pkDAAOx is widely used as a general reagent in research and bioassays of D-amino acids. Although many studies have been conducted on its purification, characterization, gene cloning, expression, and X-ray crystal structure, pkDAAOx was not shown to catalyze the oxidation of amines or even LAAOx, which belongs to the AOx protein family. 

Sources of enzyme were partially purified type II D-amino acid oxidase from porcine kidney, and the supernate from a beef kidney homogenate prepared in 2.5 volumes of 0.1 M pyrophosphate buffer (pH 8.5) containing 10 fiM FAD. The supernate obtained by centrifugation was used. 

Thus, using L-amino add oxidase from P. myxcfaciens and various amine-borane complexes or D-amino acid oxidase from porcine kidney and sodium cyanoboro-hydride, the preparation of several natural and non-natural enantiopure D- and L-amino adds was achieved, respectively [51]. In a more recent report, several P- and y-substituted a-amino adds were deracemized using D-amino add oxidase from Trigonopsis variahilis and sodium cyanoborohydride or sodium borohydride [52] (Scheme 13.20). 

Figufc 2 The comparison of the amino acid sequences of D- amino acid oxidases. The sequence of F. sofani DAO (FS) (11) is shown compared with those of the DAOs from T. Mniahlu (TV) (12) and porcine kidney (PK) (13,14). Amino acids of DAOs identical with F. sokmi DAO are shown as. ... 

---