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

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

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

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

Fluorescence emission spectra for the mutants and the wild type dephosphocoenzyme A kinase from <i>Mycobacterium tuberculosis</i> excited at 295 nm and recorded at 300–400 nm.

<p>Fluorescence emission spectra for the mutants and the wild type dephosphocoenzyme A kinase from <i>Mycobacterium tuberculosis</i> excited at 295 nm and recorded at 300–400 nm.</p

Representative profiles for mutant enzyme kinetics (by a coupled kinetic assay and ITC) and binding measurements.

<p><i>A</i>. D32N enzyme kinetics with ATP as calculated by the ITC assay. 1.4 mL of 1 µM D32N (dialyzed 12–18 hrs in 25 mM Tris buffer, pH 7.8, 150 mM NaCl, 10 mM MgCl₂, 20 mM KCl, 5% glycerol) was preincubated with 1 mM DCoA for 30 mins and was titrated against 1 mM ATP (298 µL) at 20°C. Raw data were collected for substrate heats of dilution in the buffer and integrated using the Microcal Origin 7.0 software. <i>B</i>, K14A enzyme kinetics with Dephosphocoenzyme A, as calculated by the coupled α-ketoglutarate dehydrogenase assay. <i>C</i> and <i>D</i>, titration of 1 mM DCoA against 100 µM K14A, <i>E</i> and <i>F</i>, titration of 1 mM ATP against 1 mM DCoA-saturated R144A showing that the R144A mutant shows poor binding to ATP. Raw data are shown as differential power signals in <i>C</i> and <i>E</i>. In <i>D</i> and <i>F</i>, the area under the curve produced on each injection was integrated (<i>filled squares</i>) and was plotted against the molar ratio of DCoA to enzyme binding sites using the Origin 7.0 software. The <i>solid lines</i> represent nonlinear best fits for a single-site binding model. Titrations were performed in 50 mM Tris/HCl, 150 mM NaCl, 5% glycerol, pH 7.8 at 20°C.</p

Secondary structural characterization of the mutants: Far UV CD spectra of the native CoaE and its mutants.

<p>For each mutant protein at 10 µM concentration in 10 mM phosphate buffer, pH 8.0, an average of three scans were recorded scanning from 195 nm to 260 nm.</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