Acetyltransfer Precedes Uridylyltransfer in the Formation of UDP-N-acetylglucosamine in Separable Active Sites of the Bifunctional GlmU Protein of Escherichia coli

The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788−5795], a fundamental precursor in bacterial peptidoglycan biosynthesis and the source of activated N-acetylglucosamine in lipopolysaccharide biosynthesis in Gram-negative bacteria. In the work described here, the GlmU protein and truncation variants of GlmU (N- and C-terminal) were purified and kinetically characterized for substrate specificity and reaction order. It was determined that the GlmU protein first catalyzed acetyltransfer followed by uridylyltransfer. The N-terminal portion of the enzyme was capable of only uridylyltransfer, and the C-terminus catalyzed only acetyltransfer. GlmU demonstrated a 12-fold kinetic preference (k cat/K m, 3.1 × 105 versus 2.5 × 104 L·mol-1·s-1) for acetyltransfer from acetyl-CoA to glucosamine-1-P as compared to UDP-glucosamine. No detectable uridylyltransfer from UTP to glucosamine-1-P was observed in the presence of GlmU; however, the enzyme was competent in catalyzing the formation of UDP-N-acetylglucosamine from UTP and N-acetylglucosamine-1-P (k cat/K m 1.2 × 106 L·mol-1·s-1). A two active site model for the GlmU protein was indicated both by domain dissection experiments and by assay of the bifunctional reaction. Kinetic studies demonstrated that a pre-steady-state lag in the production of UDP-N-acetylglucosamine from acetyl-CoA, UTP, and glucosamine-1-P was due to the release and accumulation of steady-state levels of the intermediate N-acetylglucosamine-1-P.