Structural and mechanistic studies on N-(2-carboxyethyl)arginine synthase |
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Authors: | Matthew E.C. Caines Christopher J. Schofield |
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Affiliation: | Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom |
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Abstract: | N2-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the β-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates d-glyceraldehyde-3-phosphate (d-G3P) and l-arginine, and are consistent with the proposed CEAS mechanism in which d-G3P binds at the active site and reacts to form an α,β-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the α-amino group of l-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the l-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative β-amino acid products. |
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Keywords: | N2-(2-Carboxyethyl)arginine synthase Thiamin diphosphate Antibiotic β-Lactam Clavulanic acid biosynthesis Penicillin resistance |
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