UDP-N-ACETYLGLUCOSAMINE ACYLTRANSFERASE OF ESCHERICHIA-COLI - THE 1STSTEP 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-h ydroxymyristoyl)GlcNAc + ACP. Using Matrex Gel Green A and heparin-aga rose, we have purified the enzyme to near homogeneity from a strain th at overproduces it 474-fold. The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with resul ts of previous radiolabeling experiments in mini-cells. The amino-term inal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA seque ncing (Coleman, J., and Raetz, C. R. H. (1988) J. Bacteriol. 170, 1268 -1274). At saturating concentrations of the second substrate, the appa rent K(m) values for UDP-GIcNAc and R-3-hydroxymyristoyl-ACP are 99 an d 1.6 muM, respectively. There is an absolute requirement for the R-3- hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds ef fectively (IC50 = 2 muM) but is inactive (<).01%) as an alternate subs trate. 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 ac yltransferase 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.