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 β‐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 P2₁ 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. Proteins 2010. © 2009 Wiley‐Liss, Inc.