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

Journal article

N(2)-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the b-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.

Authors: Caines, M; Sorensen, J; Schofield, C

• Publication status: Published
• Journal: Biochemical and biophysical research communications
• Volume: 385
• Issue: 4
• Pages: 512-517
• Publication date: 2009-08-01
• DOI: 10.1016/j.bbrc.2009.05.095
• EISSN: 1090-2104
• ISSN: 0006-291X
• Language: English
• Keywords: Binding Sites; Crystallography, X-Ray; Valerates; Argininosuccinate Lyase; Streptomyces; Argininosuccinate Synthase; Arginine; Tartrates; Protein Conformation; Multienzyme Complexes; Protein Engineering