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Glutaryl-7-aminocephalosporanic acid acylases (GA) catalyze selective cleavage of the side chain of the cephalosporins while leaving cephalosporanic nucleus intact. Glutaryl-7-aminocephalosporanic acid (GACA) is the most specific substrate whereas structurally similar natural antibiotic cephalosporin C (CephC) is not hydrolyzed by this enzyme [1]. Analysis of structural determinants of substrate discrimination can help to understand molecular mechanisms of catalysis and design new functional properties. Enzymes within Ntn-hydrolase superfamily homologous to Brevundimonas diminuta GA were identified and used to build multiple structure-based sequence alignment [2]. Recently developed bioinformatic analysis method [3, 4] was used to identify subfamily-specific positions (SSPs) – conserved only within protein subfamilies, but different between subfamilies – that seem to play important role in discrimination of substrate specificity in B. diminuta GA. These residues were used as hotspots to introduce specificity to CephC in the B. diminuta GA and the corresponding in silico library of enzyme variants containing single, double, triple or quadruple substitutions was constructed. Computer screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Consequently, GA variants were selected that significantly improve affinity to CephC by providing favorable electrostatic interactions to accommodate D-α-aminoadipic group of the substrate. The selected mutants of B. diminuta GA with improved catalytic activity towards CephC were recommended for experimental evaluation. [1] Otten et.al. (2004) Chembiochem, 5(6), 820-825 [2] Suplatov, et.al. (2011) Acta Naturae, 3(1), 93-98 [3] Suplatov et.al. (2013) J Biomol Struct Dyn., DOI: 10.1080/07391102.2012.750249 [4] http://biokinet.belozersky.msu.ru/zebra