Probing the Substrate Specificity and Intersubunit Interactions of Brevundimonas Diminuta Glutaryl Acylase with Site-Directed Mutagenesis

The Glutaryl-7-ACA acylases (GLA) are industrial enzymes widely used for the production of 7-aminocepahlosporanic acid (7-ACA)-the starting compound for manufacturing of semisynthetic cephalosporin antibiotics. Generation of mutant GLA's with increased activity and stability, capability for single-step conversion of cephalosporin C (CPC) directly to 7-ACA is a promising route to improve the current biocatalytic technologies. In this study, GLA from Brevundimonas diminuta (BrdGLA) has been rationally re-designed to produce enzyme variants with improved properties. First, sequence analysis was performed to select residues responsible for substrate specificity as hotspots to introduce the capability to bind CPC in the active site of BrdGLA. Second, BrdGLA mutants with increased activity and stability in alkaline conditions were obtained by mutating one of the surface lysine residues and replacement of the glutamine residue located in the active center by asparagine. Finally, structural analysis was used to select amino acid residues involved in formation of the quaternary structure of BrdGLA.