title: Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli.
abstract: 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 (kcat/Km, 3.1 x 10(5) versus 2.5 x 10(4) 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 (kcat/Km 1.2 x 10(6) 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Gehring
authors.forename: W J
authors.initials: WJ
authors.lastname: Lees
authors.forename: D J
authors.initials: DJ
authors.lastname: Mindiola
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: E D
authors.initials: ED
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis*
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D014537:Uridine Diphosphate N-Acetylglucosamine; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase