title: Insights into the regulatory characteristics of the mycobacterial dephosphocoenzyme A kinase: implications for the universal CoA biosynthesis pathway.
abstract: Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA).
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Guneet
authors: G
authors: Walia
authors: Avadhesha
authors: A
authors: Surolia
mesh_terms: D053898:Biosynthetic Pathways / Q000187:drug effects; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000096:biosynthesis*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D057075:Enzyme Assays; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D019163:Reducing Agents / Q000494:pharmacology; D012542:Scattering, Radiation; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D003432:Cross-Linking Reagents; D019163:Reducing Agents; D003570:Cytidine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020021:dephospho-CoA kinase; D003065:Coenzyme A