Structural and mechanistic studies on N2-(2-carboxyethyl)arginine synthase

N-2-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the beta-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 alpha,beta-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the alpha-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 beta-amino acid products. (c) 2009 Elsevier Inc. All rights reserved.