Crystal Structures of Streptococcus pneumoniaeN-Acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo Form at 2.33 Å Resolution and in Complex with UDP-N-Acetylglucosamine and Mg2+ at 1.96 Å Resolution

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33Å resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg2+ at 1.96Å resolution. The protein structure consists of an N-terminal domain with an α/β-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed β-sheet helix (LβH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed β-sheet helix domain. In the crystal, S.pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed β-sheet helix domains. UDP-N-acetylglucosamine and Mg2+ are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg2+ and the side-chain of Lys22.