Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

Abstract

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4-phosphopantetheine 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 acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 M and slightly inhibited at concentrations of 5-10 M. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3,5-ADP) suggested that it is competitive with 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 suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds 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.

Characterization of a New Type of Phosphopantetheinyl Transferase for Fatty Acid and Siderophore Synthesis in Pseudomonas aeruginosa

“…This phenomenon has first been described for AcpS of S. pneumoniae (9) and also recently for AcpS of B. subtilis (7) with B.s.-ACP. By contrast, saturation at low PCP and ArCP concentrations with increasing velocity values at higher concentrations has never been encountered with any other PPTase.…”

“…The name-giving prototype of the first group is AcpS of E. coli. PPTases of this group have a narrow substrate specificity, are about 120 aa in size, and are shown to act as homotrimers (8,9). AcpS of E. coli modifies only ACPs of FAS and type II PKS (10).…”

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

“…For instance, the ACPs from Sinorhizobium meliloti and Streptococcus pneumoniae have been overproduced and isolated from E. coli as apo-versions of ACP (27,28). On the other hand, overexpression in E. coli of the ACPs from Bacillus subtilis (17) and Pseudomonas aeruginosa (29) generated the recombinant ACP containing the 4Ј-phosphopantetheine group.…”

Characterization of Streptococcus tigurinus Small-Colony Variants Causing Prosthetic Joint Infection by Comparative Whole-Genome Analyses

“…For both S. tigurinus 2425 and 2426, we identified a mutation in the acpS gene encoding the 4=-phosphopantetheinyl transferase AcpS, which is an important enzyme in the type II fatty acid biosynthesis pathway (Table 1). Fatty acids are essential components of bacterial membrane lipids and lipopolysaccharides (33).…”