Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites

N-Acetylglucosamine-1-PO4 uridyltransferase (GlmU) is a trimeric bifunction al enzyme that catalyzes the last two sequential reactions in the de novo b iosynthetic pathway for UDP-GlcNAc. The X-ray crystal structure of Escheric hia coli GlmU in complex with UDP-GlcNAc and CoA has been determined to 2.1 Angstrom resolution and reveals a two-domain architecture that is responsi ble for these two reactions. The C-terminal domain is responsible for the C oA-dependent acetylation of Glc-1-PO4 to GlcNAc-1-PO4 and displays the long est left-handed parallel beta -helix observed to date. The acetyltransferas e active site defined by the binding site for CoA makes use of residues fro m all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO4 acetyl acceptor. The N-terminal domain catalyz es uridyl transfer from UTP to GlcNAc-1-PO4 to form the final products UDP- GlcNAc and pyrophosphate. This domain is composed of a central seven-strand ed P-sheet surrounded by six a-helices in a Rossmann fold-like topology. A Co2+ ion binds to just one of the two independent pyrophosphorylase active sites present in the crystals studied here, each of which nonetheless binds UDP-GlcNAc. The conformational changes of the enzyme and sugar nucleotide that accompany metal binding may provide a window into the structural dynam ics that accompany catalysis.