Effects of an extra Trp113Tyr substitution on yeast D-amino acid oxidase variant

Background b-Lactam antibiotics (cephalosporins and penicillins) are used in clinical practice to combat microbial infections. Commercial cephalosporins are all semi-synthetic and chemically derived from 7-aminocephalosporanic acid (7-ACA), a cephem nucleus that is traditionally produced by multi-step chemical reactions from cephalosporin C (CPC). However, the procedures are environmentally damaging, costly, and low yield [1]. The increasing annual demand for semi-synthetic cephalosporins and the concept of environmental sustainability have created a dire need to develop a ‘greener’ production of 7-ACA. 7-ACA can be enzymatically produced from CPC by a two-step process that involves D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase. Recently, we characterised the highly active and thermostable Trigonopsis variabilis DAAO (TvDAAO) variant F54Y for oxidative deamination of CPC. A single amino acid substitution at the 54th position of TvDAAO from phenylalanine (F) to tyrosine (Y) resulted in a 6-fold improvement in catalytic activity towards CPC and in thermostability after heat treatment at 55°C [2]. The potential application of the TvDAAO F54Y variant in the antibiotics industry as a simple and efficient biocatalyst was evaluated [3]. To further elucidate the structural basis of the catalytic activity and thermostability of TvDAAO F54Y, we investigated the role of the 113th amino acid residue (tryptophan, W113) that locates near the substrate binding cleft.