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Identification of critical ligand binding determinants in Mycobacterium tuberculosis adenosine-5'-phosphosulfate reductase.

ABSTRACT: Mycobacterium tuberculosis adenosine-5'-phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.

SUBMITTER: Hong JA

PROVIDER: S-EPMC2749248 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Identification of critical ligand binding determinants in Mycobacterium tuberculosis adenosine-5'-phosphosulfate reductase.

Hong Jiyoung A JA Bhave Devayani P DP Carroll Kate S KS

Journal of medicinal chemistry 20090901 17

Mycobacterium tuberculosis adenosine-5'-phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for sub ...[more]

PMID: 19678707

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Project description:Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.

Project description:Lateral gene transfer affects the evolutionary path of key genes involved in ancient metabolic traits, such as sulfate respiration, even more than previously expected. In this study, the phylogeny of the adenosine-5'-phosphosulfate (APS) reductase was analyzed. APS reductase is a key enzyme in sulfate respiration present in all sulfate-respiring prokaryotes. A newly developed PCR assay was used to amplify and sequence a fragment ( approximately 900 bp) of the APS reductase gene, apsA, from a taxonomically wide range of sulfate-reducing prokaryotes (n = 60). Comparative phylogenetic analysis of all obtained and available ApsA sequences indicated a high degree of sequence conservation in the region analyzed. However, a comparison of ApsA- and 16S rRNA-based phylogenetic trees revealed topological incongruences affecting seven members of the Syntrophobacteraceae and three members of the Nitrospinaceae, which were clearly monophyletic with gram-positive sulfate-reducing bacteria (SRB). In addition, Thermodesulfovibrio islandicus and Thermodesulfobacterium thermophilum, Thermodesulfobacterium commune, and Thermodesulfobacterium hveragerdense clearly branched off between the radiation of the delta-proteobacterial gram-negative SRB and the gram-positive SRB and not close to the root of the tree as expected from 16S rRNA phylogeny. The most parsimonious explanation for these discrepancies in tree topologies is lateral transfer of apsA genes across bacterial divisions. Similar patterns of insertions and deletions in ApsA sequences of donor and recipient lineages provide additional evidence for lateral gene transfer. From a subset of reference strains (n = 25), a fragment of the dissimilatory sulfite reductase genes (dsrAB), which have recently been proposed to have undergone multiple lateral gene transfers (M. Klein et al., J. Bacteriol. 183:6028-6035, 2001), was also amplified and sequenced. Phylogenetic comparison of DsrAB- and ApsA-based trees suggests a frequent involvement of gram-positive and thermophilic SRB in lateral gene transfer events among SRB.

Dataset Information

Identification of critical ligand binding determinants in Mycobacterium tuberculosis adenosine-5'-phosphosulfate reductase.

Publications

Identification of critical ligand binding determinants in Mycobacterium tuberculosis adenosine-5'-phosphosulfate reductase.

Similar Datasets

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