Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynth esis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phos phopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acid s and lipids. To further understand the physiological role of AcpS, we iden tified, cloned, and expressed the acpS and acpP genes of Streptococcus pneu moniae and purified both products to homogeneity. Both acpS and acpP form o perons with the genes whose functions are required for other cellular metab olism. The acpS gene complements an Escherichia coli mutant defective in th e production of AcpS and appears to be essential for the growth of S. pneum oniae. Gel filtration and cross-linking analyses establish that purified Ac pS exists as a homotrimer. AcpS activity was significantly stimulated by ap o-ACP at concentrations over 10 mu M and slightly inhibited at concentratio ns of 5-10 mu M. Double reciprocal analysis of initial velocities of AcpS a t various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibiti on kinetics of the product (3',5'-ADP) suggested that it is competitive wit h respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results sug gest that AcpS may be allosterically regulated by apo ACP and probably proc eeds by an ordered reaction mechanism with the first formation of the AcpS- apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.