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Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis.

ABSTRACT: Dephosphocoenzyme A kinase performs the transfer of the ?-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 Mg(2+) 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.

SUBMITTER: Walia G

PROVIDER: S-EPMC3019153 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis.

Walia Guneet G Gajendar Komatireddy K Surolia Avadhesha A

PloS one 20110111 1

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 ...[more]

PMID: 21264299

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Project description:The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

Dataset Information

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis.

Publications

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis.

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