The bifunctional bacterial enzymeN-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against β-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. TheS. pneumoniae GlmU molecule is organized in two separate domains connected via a long α-helical linker and associates as a trimer, with the 50-Å-long left-handed β-helix (LβH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LβH core and exchanged with the β-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LβH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.