Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

Abstract

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

Figure 1.

E. coli GlmU in complex with substrates. (A) Native trimeric structure of GlmU. The three acetyltransferase active sites associated with the left-handed β-helical domain (shown in complex with CoA and GlcN-1-PO₄) are depicted at the bottom. The three pyrophosphorylase active sites (in complex with UDP-GlcNAc and Co²⁺) are at the top. For the proximal acetyltransferase active site, the A and B subunits are colored blue and green, respectively, with the insertion loop of subunit B in red. (B) Acetyltransferase active site viewed from the same direction as in A. The inserted loop of subunit B is shown (red). (C) Acetyltransferase active-site interactions with the pantetheinyl group of acetyl-CoA. Close hydrophilic interactions are indicated by dotted segments. The interactions formed to CoA are similar. (D) Acetyltransferase active-site interactions with GlcNAc-1-PO₄. The interactions formed to GlcN-1-PO₄ are similar. (E) Stereoview superposition of GlmU acetyltransferase active-site complexes; acetyl-CoA/SO₄ (blue bonds), CoA/GlcN-1-PO₄ (yellow), and desulpho-CoA/GlcNAc-1-PO₄ (green). The residue labeling refers to the A or B subunits of a single acetyltransferase active site. The superimposed coenzyme and phosphosugar molecules are labeled for acetyl-CoA and GlcNAc-1-PO₄, respectively. Residues 386B and 387B from the insertion loop are visible. Figure prepared with PyMOL (DeLano 2002).