Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway.
(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.
Iqbal
Aman
A
University of Oxford, United Kingdom.
Arunlanantham
Haren
H
Brown
Tom
T
Chowdhury
Rasheduzzaman
R
Clifton
Ian J
IJ
Kershaw
Nadia J
NJ
Hewitson
Kirsty S
KS
McDonough
Michael A
MA
Schofield
Christopher J
CJ
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*
D019818:Clavulanic Acid; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases