title: Structural and mechanistic studies on N(2)-(2-carboxyethyl)arginine synthase.
abstract: 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.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX13TA, United Kingdom.
authors.forename: Matthew E C
authors.initials: ME
authors.lastname: Caines
authors.forename: John L
authors.initials: JL
authors.lastname: Sorensen
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D001120:Arginine / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D013302:Streptomyces / Q000201:enzymology*; D013644:Tartrates / Q000737:chemistry / Q000378:metabolism; D014631:Valerates / Q000737:chemistry / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D013644:Tartrates; D014631:Valerates; C019726:arginine synthetase system; D001120:Arginine; D001123:Argininosuccinate Lyase; D001124:Argininosuccinate Synthase; C029768:tartaric acid