Targeting the biosynthetic pathway of
Coenzyme A (
CoA) for
drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final
enzymes of
CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final
enzyme,
dephosphocoenzyme A kinase (CoaE) as a potential
drug target.
METHODOLOGY/PRINCIPAL FINDINGS: We report here a complete biochemical and biophysical characterization of the M.
tuberculosis CoaE, an
enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a
dephosphocoenzyme A kinase with its substrates,
dephosphocoenzyme A and
ATP; its product,
CoA and an intrinsic yet novel inhibitor,
CTP, which helps modulate the
enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial
enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the
enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and
ATP following in tow. Kinetic and ITC experiments demonstrate that though
CTP binds strongly to the
enzyme, it is unable to participate in DCoA phosphorylation. We report that
CTP actually inhibits the
enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate
kinases,
deoxycytidine kinases, and cytidylate
kinases as close homologs. Docking of DCoA and
CTP to CoaE shows that both
ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown
Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length
enzyme.
CONCLUSIONS/SIGNIFICANCE: In view of the evidence presented, it is imperative to assign a greater role to the last
enzyme of
Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.