Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

Proteins. 2010 May 1;78(6):1398-407. doi: 10.1002/prot.22653.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl Coenzyme A / metabolism
  • Acetyltransferases / chemistry*
  • Binding Sites
  • Clavulanic Acid / biosynthesis*
  • Clavulanic Acid / chemistry
  • Crystallography, X-Ray
  • Metabolic Networks and Pathways*
  • Models, Molecular
  • Protein Denaturation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Spectrometry, Mass, Electrospray Ionization*
  • Streptomyces / enzymology*

Substances

  • Clavulanic Acid
  • Acetyl Coenzyme A
  • Acetyltransferases