Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5–10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE

Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

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)

The Role of UPF0157 in the Folding of M. tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

BACKGROUND: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

The on-column glutaraldehyde crosslinking of the mycobacterial CoaE.

<p>The 8% SDS-PAGE gel picture shows the CoaE protein loaded on the column (Load); the flowthrough from the column during loading (FT), the eluate during the wash steps (WI, WII and WIII); the molecular weight marker (M) and the elution aliquots (E1 and E2).</p

Effects of CTP on CoaE enzymatic activity.

<p>(A), Histogram depicting the percent decrease in CoaE activity in the presence of CTP, with the activity of CoaE in the presence of ATP (1 mM) and DCoA (0.5 mM) treated as 100% (Lane 1). Lanes 2–6 show the percent activity of CoaE in the presence of different concentrations of CTP; Lane 2, 50 µM CTP; Lane 3, 100 µM CTP; Lane 4, 250 µM CTP; Lane 5, 500 µM CTP; Lane 6, 1 mM CTP. (B), CoaE reaction velocity plotted versus leading substrate, DCoA concentrations (10–640 µM) in the presence and absence of CTP (1 mM), measured using the coupled PK-LDH spectrophotometric assay.</p

Schematic depicting the dynamic interplay between the enzyme's leading substrate, DCoA and the cellular metabolite, CTP, in regulating the activity of the last enzyme of mycobacterial CoA biosynthesis.

<p>The enzyme is sequestered in a trimeric state that renders it inactive, potentially due to spatial constraints imposed on the mobile lid and CoA domains. DCoA, upon binding, induces monomerization, releasing these constraints and inducing conformational changes in the enzyme, allowing the phosphate donor, ATP, to bind and catalysis to occur. CTP, on the other hand, prevents DCoA from binding the enzyme by virtue of occupying a similar binding site on the enzyme, thereby preventing the oligomeric re-equilibration, reflected in a mere 16% active CoaE in the presence of 1 mM CTP. Thus the mycobacterial cell employs regulation at the last enzyme of CoA biosynthesis via two co-acting mechanisms.</p

Inhibition kinetics.

<p>(A), Measurement of the specific activity of CoaE as a function of enzyme concentration. Freshly prepared CoaE was diluted in buffer alone or was pre-saturated with 1 mM CTP and diluted in buffer+CTP to a range of concentrations (10 nM–10 µM) and activity was measured at 0.5 mM DCoA. (B), Dixon plot (1/V vs. [CTP]) illustrating the inhibition of the mycobacterial dephosphocoenzyme A kinase by CTP (Ki = 34 µM), at three different concentrations of the leading substrate, DCoA.</p

Determination of the oligomeric status of CoaE by size exclusion chromatography.

<p>(A), Each individual reaction mixture, incubated for 30 mins, contained CoaE alone; CoaE+1 mM DCoA; CoaE+1 mM ATP+1 mM MgCl₂ was loaded on a Superdex S-200 column. (B), Each individual reaction mixture, incubated for 30 mins, contained CoaE+1 mM DCoA; CoaE+1 mM CTP (incubated for 30 mins) + DCoA (incubated for another 30 mins); CoaE+ 1 mM Malonyl-CoA. Peak 1: Trimeric CoaE (M_r ∼140,000±2900 Da), Peak 2: Monomeric CoaE (M_r ∼47,000±15 Da) (Molecular masses expressed as mean ± S.D. of five independent experiments).</p

Limited proteolysis of the mycobacterial CoaE studied by tryptic cleavage at various time points and separation of the digestion products on an 8% SDS-PAGE gel.

<p>(A), tryptic digestion fragmentation pattern for CoaE. (B), protection afforded by the leading substrate, DCoA, on the mycobacterial enzyme during proteolysis. (C), fragmentation pattern of CoaE digestion in the presence of CTP. (D), Comparison of the cleavage patterns of CoaE alone and that in the presence of DCoA. (-Tryp) denotes the enzyme aliquot in the absence of the protease.</p