Journal Article

Cloning and Expression in <i>Escherichia coli</i> of the <span class="smallCaps">d</span>-Aspartate Oxidase Gene from the Yeast <i>Cryptococcus humicola</i> and Characterization of the Recombinant Enzyme

Shouji Takahashi, Toshiyuki Takahashi, Yoshio Kera, Ryuji Matsunaga, Hiroo Shibuya and Ryo-hei Yamada

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 135, issue 4, pages 533-540
Published in print April 2004 | ISSN: 0021-924X
Published online April 2004 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/jb/mvh068
Cloning and Expression in Escherichia coli of the d-Aspartate Oxidase Gene from the Yeast Cryptococcus humicola and Characterization of the Recombinant Enzyme

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The d-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to d-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in d-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4–33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of d-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.

Keywords: Cryptococcus humicola, d-aspartate oxidase, flavoenzyme, gene expression, yeast.; CBB, Coomassie Brilliant Blue; ChDDO, d-aspartate oxidase from Cryptococcus humicola UJ1; DAO, d-amino acid oxidase; DDO, d-aspartate oxidase; GABA, γ -aminobutyrate; IPTG, isopropyl-β-d-thiogalactopyranoside; LB, Luria-Bertani; pigDAO, pig kidney DAO; PTS1, peroxisomal targeting signal 1; RACE, rapid amplification of cDNA ends; RgDAO, d-amino acid oxidase from Rhodotorula gracilis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSC, standard saline citrate; TB, Terrific Broth.

Journal Article.  5471 words.  Illustrated.

Subjects: Biochemistry

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