The Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Synthase III Activity Is Inhibited by Phosphorylation on a Single Threonine ResidueS
Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C 56-64 -meromycolyl chain and a C 24-26 -fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I...
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Veröffentlicht in: | The Journal of biological chemistry 2009-03, Vol.284 (10), p.6414-6424 |
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Zusammenfassung: | Mycolic acids are hallmark features of the
Mycobacterium
tuberculosis
cell wall. They are synthesized by the condensation of two
fatty acids, a C
56-64
-meromycolyl chain and a
C
24-26
-fatty acyl chain. Meromycolates are produced via the
combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The
β-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I
and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with
malonyl-ACP. Because mtFabH represents a potential regulatory key point of the
mycolic acid pathway, we investigated the hypothesis that phosphorylation of
mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein
kinases (STPKs) has recently emerged as a major physiological mechanism of
regulation in prokaryotes. We demonstrate here that mtFabH was efficiently
phosphorylated
in vitro
by several mycobacterial STPKs, particularly
by PknF and PknA, as well as
in vivo
in mycobacteria. Analysis of the
phosphoamino acid content indicated that mtFabH was phosphorylated exclusively
on threonine residues. Mass spectrometry analyses using liquid
chromatography-electrospray ionization/tandem mass spectrometry identified
Thr
45
as the unique phosphoacceptor. This was further supported by
complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant.
Mapping Thr
45
on the crystal structure of mtFabH illustrates that
this residue is located at the entrance of the substrate channel, suggesting
that the phosphate group may alter accessibility of the substrate and thus
affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic
constitutive phosphorylation, exhibited markedly decreased transacylation,
malonyl-AcpM decarboxylation, and condensing activities compared with the
wild-type protein or the T45A mutant. Together, these findings not only
represent the first demonstration of phosphorylation of a β-ketoacyl-ACP
synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits
its enzymatic activity, which may have important consequences in regulating
mycolic acid biosynthesis. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M806537200 |