Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues |
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Authors: | Oh Bora Kim Kyunggon Park Jungeun Yoon Jongchul Han Dohyun Kim Youngsoo |
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Institution: | Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, Republic of Korea. |
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Abstract: | The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA. |
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Keywords: | Penicillin G acylase Cephalosporin acylase Protein engineering N-terminal hydrolase Divergent evolution Structure-based enzyme design |
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