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

Abstract

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.

Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

“…Our other mutational studies have shown that conserved residues of the Walker A/P-loop motif are critical for LpxK function, as has been observed for other P-loop kinases (39,40). Mutation of three P-loop glycine residues inhibited activity significantly, especially those nearer the conserved P-loop lysine.…”

“…First, only LpxK incubated with ATP/Mg 2+ crystallized with ADP and Mg 2+ in the active site, indicating that ATPase activity may have been achieved in the absence of DSMP during crystallization. Second, in other P-loop kinases, the Walker A hydroxyl-containing residue (T52 in LpxK), which follows the P-loop lysine, is often found to be directly involved in Mg 2+ binding (19)(20)(21)(22) and is sometimes assisted by a Walker B carboxylate (19,20,31,40). The precatalytic conformation of ATP/Mg 2+ -bound LpxK may be well-represented by the GTPγS/Mg 2+ -bound model of LpxK's closest structural homolog HypB, in which the γ-phosphate occupies an analogous position to the Mg 2+ ion of the LpxK structure (Fig.…”

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.…”

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

“…Utilizing the primers, C235S_Fwd: 5′ –GATCGCGTCCGGGCATAAGGCCTTG- 3′ and C235S_Rev: 5′ –ATGCCCGGACGCGATCTTTAG- 3′ and the set, C368S_Fwd: 5′ –GAAGACTATTTGACGGTCAAGTCTGACGCCGACAG- 3′ and C368S_Rev: 5′-GTCGGCGCGCCTGTCGGCGTCAGACTTGACC- 3′ , the CoaE cysteine mutants, C235S and C368S, were PCR-amplified from the mycobacterial genomic DNA using the same conditions as the wild type protein described in [17] . The cysteine mutant proteins were expressed and purified like wild type CoaE [17] , [19] .…”