title: Activation of a peroxisomal Pichia pastoris D-amino acid oxidase, which uses d-alanine as a preferred substrate, depends on pyruvate carboxylase.
abstract: d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses d-alanine as a substrate, and Dao2p, which uses d-aspartate as a preferred substrate. Dao1p has a molecular mass of 38.2 kDa and a pH optimum of 9.6. This enzyme was localized to peroxisomes, albeit a typical peroxisomal targeting signal is lacking. Interestingly, P. pastoris mutant strains, defective in the enzyme pyruvate carboxylase, showed a pronounced growth defect on d-alanine, concomitant with a significant reduction in Dao1p activity relative to the wild-type control. This indicates that pyruvate carboxylase functions in import and/or activation of P. pastoris Dao1p.
authors.affiliation: Molecular Cell Biology, University of Groningen, AA Haren, The Netherlands.
authors.forename: Sandra H
authors.initials: SH
authors.lastname: Klompmaker
authors.forename: Aysun
authors.initials: A
authors.lastname: Kilic
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Baerends
authors.forename: Marten
authors.initials: M
authors.lastname: Veenhuis
authors.forename: Ida J
authors.initials: IJ
authors.lastname: van der Klei
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D003470:Culture Media / Q000737:chemistry; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020675:Peroxisomes / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology* / Q000254:growth & development; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003470:Culture Media; D003605:D-Amino-Acid Oxidase; D011766:Pyruvate Carboxylase; D000409:Alanine