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J. Biol. Chem., Vol. 275, Issue 40, 30864-30872, October 6, 2000
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From the Lilly Research Laboratories, the Eli Lilly and Company,
Indianapolis, Indiana 46285
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 µM and slightly inhibited
at concentrations of 5-10 µM. 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.
Biochemical and Molecular Analyses of the Streptococcus
pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential
for Fatty Acid Biosynthesis*
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: The Lilly Research
Laboratories, Infectious Diseases Research, Eli Lilly and Company, DC
0438, Indianapolis, Indiana 46285.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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