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.
Highlights
Coenzyme A is an essential metabolic cofactor which participates in more than 9% of all enzymatic reactions in the cellular milieu [1]
Coenzyme A is synthesized from its cellular precursors, pantothenate, cysteine and ATP in a series of five steps, initiated by pantothenate kinase (CoaA) which phosphorylates pantothenate to generate 49-phosphopantothenate. The latter is fused with a cysteine moiety by CoaB (49phosphopantothenoylcsyteine synthetase) to generate 49-phosphopantothenoylcysteine which is decarboxylated by CoaC (49phosphopantothenoylcysteine decarboxylase). 49-phosphopanthetheine, the product of the CoaC reaction is adenylated by 49phosphoadenylyltransferase (CoaD) to generate 49-dephosphocoenzyme A
These enzymes share several common structural motifs despite having a negligible sequence similarity amongst each other. The chief amongst these is the Ploop and the overall three-dimensional fold comprising of three domains. The latter together form the nucleotide-binding fold of these P-loop containing proteins; the five-stranded parallel b-sheet which forms the nucleotide-binding domain flanked on both sides by the a-helical substrate binding domain and the a-helical Lid domain
Summary
Coenzyme A is an essential metabolic cofactor which participates in more than 9% of all enzymatic reactions in the cellular milieu [1]. Coenzyme A is synthesized from its cellular precursors, pantothenate, cysteine and ATP in a series of five steps, initiated by pantothenate kinase (CoaA) which phosphorylates pantothenate to generate 49-phosphopantothenate. The latter is fused with a cysteine moiety by CoaB (49phosphopantothenoylcsyteine synthetase) to generate 49-phosphopantothenoylcysteine which is decarboxylated by CoaC (49phosphopantothenoylcysteine decarboxylase). CoaEs belong to the family of nucleotide and nucleoside kinases, which are members of the structural superfamily of NTP (nucleoside triphosphate) hydrolases according to the SCOP hierarchy [2] These enzymes share several common structural motifs despite having a negligible sequence similarity amongst each other. The E. coli enzyme is a crystallographic trimer while the Thermus and Haemophilus enzymes were monomeric in the crystal state
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