Abstract
Free D-aspartate has been recently found in a large amount in several animals and plays an important role in various physiological processes. The D-amino acid is considered to be synthesized and degraded mainly by aspartate racemase (AspR) and D-aspartate oxidase (DDO), respectively, in eukaryote. However, the synthetic pathway of the amino acid in eukaryote and the structure and physiological function of DDO in microorganisms remained to be elucidated. We thus purified and characterized AspR from the bivalve Scapharca broughtonii and cloned the encoding gene. The first eukaryotic AspR is the first example of pyridoxal 5'-phosphate (PLP)-dependent AspR and belongs to the fold-type II PLP enzyme family, and had unique characteristics, including the regulation of its activity by nucleotides. We next cloned the first microbial DDO gene from the yeast Cryptococcus humicola, analyzed its expression profile and constructed DDO gene-disrupted strain. The primary structure contains several common features conserved among DDO and D-amino acid oxidase (DAO), and showed a more close relation to fungal DAO. The DDO expression profile and the disrupted strain showed its role in not only the utilization of but also the protection from acidic D-amino acids for cell growth.
