UDP-N-ACETYLGLUCOSAMINE ACYLTRANSFERASE OF ESCHERICHIA-COLI - THE 1ST STEP OF ENDOTOXIN BIOSYNTHESIS IS THERMODYNAMICALLY UNFAVORABLE

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 K(m) values for UDP-GIcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 muM, respectively. There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 muM) but is inactive (<).01%) as an alternate substrate. The most remarkable feature of the reaction is its unfavorable equilibrium constant, K(eq) congruent-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.