Open AccessActivation of a peroxisomal Pichia pastoris d ‐amino acid oxidase, which uses d ‐alanine as a preferred substrate, depends on pyruvate carboxylase
Author(s) -
Klompmaker Sandra H.,
Kilic Aysun,
Baerends Richard J.,
Veenhuis Marten,
Van Der Klei Ida J.
Publication year - 2010
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2010.00647.x
Subject(s) - pichia pastoris , biochemistry , pyruvate carboxylase , peroxisome , alanine , biology , oxidative deamination , amino acid , d amino acid oxidase , oxidase test , substrate (aquarium) , enzyme , ecology , gene , recombinant dna
d ‐Amino acid oxidase (DAO) is an important flavo‐enzyme that catalyzes the oxidative deamination of d ‐amino acids into the corresponding α‐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.