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

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

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

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

Homology modeled structure of the active site.

<p>The mycobacterial dephosphocoenzyme A kinase has been homology modeled and the leading substrate, DCoA has been docked at the active site. Protein residues are shown as green ball-and-stick models and the bound DCoA in cyan. The figure was generated in PyMol.</p

Multiple Sequence Alignment of the protein sequences of dephosphocoenzyme A kinases from <i>Escherichia coli</i> (UniProtKB ID P0A619), <i>Thermus thermophilus</i> (UniProtKB ID Q56416), <i>Haemophilus influenzae</i> (UniProtKB ID P44920), <i>Mycobacterium tuberculosis</i> (UniProtKB ID P63826) and the mammalian coenzyme A synthase (UniProtKB ID Q13057) carried out by the ClustalW algorithm.

<p>For purposes of clarity, only the CoaE domain of the mycobacterial and human enzymes have been shown. The residues chosen for mutagenesis have been highlighted and colored in red. Completely conserved residues are starred.</p

Thermodynamic parameters of the binding of the substrates, DCoA and ATP to the wild type and mutant mycobacterial dephosphocoenzyme A kinases at pH 7.8.

<p>Bracketed ligands next to the enzymes refer to the substrate against which the enzyme was pre-saturated before the titration. Values of ΔH and ΔS are in calM⁻¹ and calM⁻¹K⁻¹ respectively. Values are the mean of five individual experiments. K_a is the binding constant determined by ITC and its values are in M⁻¹.</p

Effects of the mutagenesis on the kinetic parameters of the mycobacterial dephosphocoenzyme A kinase.

<p>The kinetic values depicted are a mean of values ±S.E. of two independent experiments performed in duplicates. A. Comparison of the Km values of the mutants for DCoA with that of the WT, B. Comparison of the Km values of the mutants for ATP with that of the WT. WT-Mg and D32N-Mg entries refer to the Km values obtained in experiments carried out in the absence of magnesium.</p