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 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 microm and slightly inhibited at concentrations of 5-10 microm. 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.
McAllister
K A
KA
Lilly Research Laboratories, the Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Peery
R B
RB
Meier
T I
TI
Fischl
A S
AS
Zhao
G
G
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon; D003065:Coenzyme A / Q000494:pharmacology; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis*; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D009876:Operon; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D014461:Ultracentrifugation
C101898:AcpP protein, Streptomyces; D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D003062:Codon; D003432:Cross-Linking Reagents; D005227:Fatty Acids; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D003065:Coenzyme A