Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG.
MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.
Chen
Lan
L
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
Men
Hongbin
H
Ha
Sha
S
Ye
Xiang-Yang
XY
Brunner
Livia
L
Hu
Yanan
Y
Walker
Suzanne
S
D001425:Bacterial Outer Membrane Proteins; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D016695:Glycosyltransferases / Q000378:metabolism; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D009005:Monosaccharides / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis; D013379:Substrate Specificity; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D008055:Lipids; D008565:Membrane Proteins; D009005:Monosaccharides; D009842:Oligopeptides; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C438940:lipid I; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase