Biochemistry
Article
inhA, a gene encoding a target for isoniazid and ethionamide in
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ecACP binding. R62, K63, and R66 all lie on helix α3, and
modeling studies suggest that the interaction of ecACP with
this basic patch results in delivery of the substrate to the active
site of the adjacent monomer. Thus, ecACP binding at this site
provides a mechanism that allows ecACP to interact with both
monomers at the same time. This basic patch is also adjacent to
loop 1 on the adjacent monomer which includes M269 (Figure
4B). It is thus plausible that ecACP binding to ecFabB causes
M269 to move closer to M137 which subsequently causes the
helices to flex.
This model is consistent with the absolute requirement of
KasA and ecFabB for ACP since the methionine residues that
are responsible for the hinge movement are conserved in
ecFabB and KasA but not in ecFabF. This suggests that ecFabF
can freely adopt open and closed conformations irrespective of
ecACP binding. Thus, for ecFabF the Km values are simply a
consequence of the interaction of substrates with an individual
subunit, whereas in KasA and ecFabB binding to one subunit
causes coupled motions in the adjacent monomer. For ecFabF
we observe that a 14-residue peptide derived from ACP α-helix
2 can efficiently replace ecACP in the condensation reaction.
However, remote interactions between ACP and the KASI
enzymes must play a critical role in altering the conformation of
the adjacent subunit since our peptide substrate mimics of ACP
cannot replace the full length carrier protein in the reactions
catalyzed by KasA or ecFabB. Finally, when palmitoyl-CoA is
used as the donor for the KasA reaction, substrate inhibition is
observed at concentrations above ∼3 μM. This again stresses
the importance of AcpM for the reaction catalyzed by this
enzyme and also suggests that KasA would be unable to
substitute for the loss of mtFabH, supporting the relevance of
the latter enzyme in ongoing drug discovery efforts.47,50,51
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: (631) 632-7907. Fax: (631) 632-7960. E-mail: peter.
Present Addresses
†WGS, 3907 Prince St., Flushing, NY 11354.
‡GlaxoSmithKline, 250 S. Collegeville Rd., Collegeville, PA
19426.
Funding
This work was funded by NIH Grant AI044639.
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ABBREVIATIONS
■
KAS, β-ketoacyl-acyl carrier protein synthase; PPant, phospho-
pantetheine; ACP, acyl carrier protein; AcpM, M. tuberculosis;
ecACP, E. coli; PanK, pantothenate kinase; AcpS, holo-ACP
synthase; MalPPant, malonyl-phosphopantetheine; PalmPPant,
palmitoyl-phosphopantetheine.
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dx.doi.org/10.1021/bi201199x|Biochemistry 2011, 50, 10678−10686