Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway.
The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution. The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.
Caines
Matthew E C
ME
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, United Kingdom.
Elkins
Jonathan M
JM
Hewitson
Kirsty S
KS
Schofield
Christopher J
CJ
D000596:Amino Acids / Q000737:chemistry; D001123:Argininosuccinate Lyase / Q000737:chemistry*; D001124:Argininosuccinate Synthase / Q000737:chemistry*; D001665:Binding Sites; D002850:Chromatography, Gel; D019818:Clavulanic Acid / Q000096:biosynthesis / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D008274:Magnesium / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry*; D013302:Streptomyces / Q000201:enzymology
D000596:Amino Acids; D009097:Multienzyme Complexes; C019726:arginine synthetase system; D019818:Clavulanic Acid; D001123:Argininosuccinate Lyase; D001124:Argininosuccinate Synthase; D008274:Magnesium