PMID:8366124
| Citation |
Anderson, MS, Bull, HG, Galloway, SM, Kelly, TM, Mohan, S, Radika, K and Raetz, CR (1993) UDP-N-acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin biosynthesis is thermodynamically unfavorable. J. Biol. Chem. 268:19858-65 |
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| Abstract |
UDP-N-acetylglucosamine acyltransferase of Escherichia coli catalyzes the reaction, UDP-GlcNAc + R-3-hydroxymyristoyl-ACP--> UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + ACP. Using Matrex Gel Green A and heparin-agarose, we have purified the enzyme to near homogeneity from a strain that overproduces it 474-fold. The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with results of previous radiolabeling experiments in mini-cells. The amino-terminal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA sequencing (Coleman, J., and Raetz, C. R. H. (1988) J. Bacteriol. 170, 1268-1274). At saturating concentrations of the second substrate, the apparent Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 microM, respectively. There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 microM) but is inactive (< 0.01%) as an alternate substrate. The most remarkable feature of the reaction is its unfavorable equilibrium constant, Keq approximately equal to 0.01, which is not predicted by model S-->O acyl transfer reactions. Thus, although UDP-GlcNAc acyltransferase catalyzes the first unique step of lipid A biosynthesis, it is the second enzyme (the deacetylase) that commits the substrates to this pathway. The specific activity of the deacetylase is elevated approximately 5-fold when lipid A synthesis is inhibited. |
| Links | |
| Keywords |
Acyltransferases/isolation & purification; Acyltransferases/metabolism; Chromatography, Affinity; Chromatography, Gel; Endotoxins/biosynthesis; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; Lipid A/biosynthesis; Molecular Weight; Peptidoglycan/biosynthesis; Substrate Specificity; Thermodynamics |
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