title: Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway.
abstract: (3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A beta-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2(1) space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the "second" CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins.
authors.affiliation: University of Oxford, United Kingdom.
authors.forename: Aman
authors.initials: A
authors.lastname: Iqbal
authors.forename: Haren
authors.initials: H
authors.lastname: Arunlanantham
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.forename: Rasheduzzaman
authors.initials: R
authors.lastname: Chowdhury
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: Nadia J
authors.initials: NJ
authors.lastname: Kershaw
authors.forename: Kirsty S
authors.initials: KS
authors.lastname: Hewitson
authors.forename: Michael A
authors.initials: MA
authors.lastname: McDonough
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry*; D001665:Binding Sites; D019818:Clavulanic Acid / Q000096:biosynthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D019818:Clavulanic Acid; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases