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).

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

“…35−37 These studies, together with the availability of crystal structures of a number of CoA pathway enzymes for use in structure-guided drug design, provided further support for investigating the CoA pathway as an attractive source of new TB drug targets, encouraging the development of inhibitors against various pathway enzymes. 9,13,38−41 However, despite resource-intensive efforts that led to the identification of potent inhibitors of Mtb PanC and PanK enzymes, these molecules failed to translate into lead compounds with significant whole-cell activity. 13,42,43 Using cKD mutants of Mtb as tools to assess target vulnerability and the target selectivity of small-molecule inhibitors in whole Mtb cells, we 21 and others 43 concluded that these failures might be explained, at least in part, by the relative invulnerability of Mtb to depletion of PanC and PanK.…”

Enzyme catalysis prior to aromatic residues: reverse engineering of a dephosphoCoA kinase

“…The wild type variant of DPCK performs phosphotransferase activity from ATP to dCoA where dCoA acts as the leading substrate and ATP does not get hydrolysed in its absence (30). The unliganded DPCK is a well folded protein with high α-helical content, its domain movements upon ATP binding play a crucial role during catalysis (28).…”

“…The unliganded DPCK is a well folded protein with high α-helical content, its domain movements upon ATP binding play a crucial role during catalysis (28). None of the aromatic amino acid residues has been reported essential for the ligand binding and catalysis (30).…”

Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea

“…All previously characterized DPCKs are considered to be ATP dependent, although direct experimental evidence is limited. In bacteria, DPCK enzymes from Thermus thermophilus HB8 (31), Mycobacterium tuberculosis (32–34), Streptomyces peucetius ATCC 27952 (35), and E. coli K-12 (36) have been characterized.…”