Identification and expression a D-amino acid dehydrogenase gene from Pseudomonas fluorescens TM5-2

Abstract:

In the previous study we have isolated DNA fragment containing an alanine racemase gene (dadX) from Pseudomonas fluorescens TM5-2. Adjacent to dadX one ORF similar to a putative glycine/D-amino acid oxidase gene have been found. The same gene organization is found in several Pseudomonas species. Here, author would characterize this ORF to determine what kind of enzyme this gene encodes. DNA fragment containing gene encoding putative glycine/D-amino acid oxidase was cloned into the expression vector. Firstly oxidase activity in cell lysates prepared from the recombinant cells was measured, however, neither glycine nor D-alanine were oxidized judging from hydrogen peroxide formation. Secondly when the amino acid sequence deduced from the oxidase gene was compared to dye-linked D-amino acid dehydrogenases, all the important residues including FAD-binding motif were conserved. This gene was transformed and checked on TTC plate, it showed some activities of D-amino acid dehydrogenase. D-amino acid dehydrogenase activity was also detected when D-alanine and DCIP were used. The best substrate of this enzyme is D-histdine, which is different from some reports. Author will be in progress to purify the dehydrogenase and determine enzyme characteristics.