Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg2+ at 1.96 A resolution

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential ba cterial enzyme with both an acetyltransferase and a uridyltransferase activ ity which have been mapped to the C-terminal and N-terminal domains, respec tively. GlmU performs the last two steps in the synthesis of UDP-N-acetylgl ucosamine (UDP-GlcNAc), which is an essential precursor in both the peptido glycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensi onal structure would provide a basis for rational drug design. We have dete rmined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 Angstrom resolution, and in complex with UDP-N-acetyl gluc osamine and the essential co-factor Mg2+ at 1.96 Angstrom resolution. The p rotein structure consists of an N-terminal domain with an alpha/beta -fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta -sheet helix (L betaH) domain. An insertion loop c ontaining the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes fro m the left-handed beta -sheet helix domain. Ln the crystal, S. pneumoniae G lmU forms exact trimers, mainly through contacts between left-handed beta - sheet helix domains. UDP-N-acetylglucosamine and Mg2+ are bound at the urid yltransferase active site, which is in a closed form. We propose a uridyltr ansferase 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. (C) 2001 Academic Press.