Acceptor substrate selectivity and kinetic mechanism of Bacillus subtilis TagA.
Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.
Zhang
Yu-Hui
YH
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ginsberg
Cynthia
C
Yuan
Yanqiu
Y
Walker
Suzanne
S
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013682:Teichoic Acids / Q000378:metabolism; D014530:Uridine Diphosphate / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000378:metabolism
D001426:Bacterial Proteins; D008074:Lipoproteins; D008670:Metals; D009005:Monosaccharides; D009842:Oligopeptides; C092406:TagA protein, bacteria; D013682:Teichoic Acids; D014539:Uridine Diphosphate Sugars; C438940:lipid I; C059191:uridine diphosphate N-acetylmannosamine; D014530:Uridine Diphosphate