Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae

Abstract

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.

Figure 1. Phylogenetic analysis of Rv1647

(A) The consensus neighbour-joining tree drawn in MEGA retaining branches with >50% support in an interior-branch test of phylogeny is shown. Rv1647 and ML1399 are indicated as black squares; cyclases characterized biochemically are indicated by filled circles and the empty square indicates that the cyclase (Rv0891c) has been found to be an adenylate cyclase (A. R. Shenoy and S. S. Visweswariah, unpublished work). Abbreviations for the names of organisms are: A.cyl, Anabaena cylindrical; B.jap, Bradyrhizobium japonicum; B.liq, Brevibacterium liquifaciens; C.alb, Candida albicans; C.fam, Canis familiaris; H.sap, Homo sapiens; L.don, Leishmania donovanii; L.int, Leptospira interrogans; M.ace, Methanosarcina acetivorans; M.kan, Methanopyrus kandleri; M.lep, M. leprae; M.mus, Mus musculus; M.tub, M. tuberculosis; N.cra, Neurospora crassa; R.nor, Rattus norvegicus; S.cer, Saccharomyces cerevisiae; S.coe, Streptomyces coelicolor; Syne, Synechocystis sp.; T.bru, Trypanosoma brucei.

Figure 2. Sequence analysis of Rv1647, its orthologues and genome neighbours

(A) Multiple sequence alignment of the catalytic domains of Rv1647 and its orthologues from related mycobacteria and the mammalian adenylate cyclase C1 and C2 domains. C.fam, C. familiaris; R.nor, R. norvegicus; M.tub, M. tuberculosis; M.bov, M. bovis; M.avi, M. avium subsp. paratuberculosis; M.lep, M. leprae; M.sme, M. smegmatis. ACVC1 and ACIIC2 stand for the C1 domain of the canine type V and C2 domain of the rat type II adenylate cyclase respectively. Metal co-ordinating aspartic acids are indicated by ⇓, substrate specifying residues are indicated by ∇ and the transition state stabilizing asparagine and arginine residues are indicated by V. (B) Schematic alignment output of the nucleotide sequences of the region flanking Rv1647 from related mycobacteria whose genomes have been sequenced. Arrows indicate the genes from 5′ to 3′ direction and the M. tuberculosis H37Rv gene names are indicated on top. A bar with numbers in kb is given as a ruler. Solid lines indicate bit scores of >200 and slashed lines indicate score of <40. Notice the absence of the gene from the PE family in M. avium and M. leprae.

Figure 3. Purification and enzymatic activity of Rv1647

Coomassie-stained SDS/polyacrylamide gel of the purified Rv1647 catalytic domain protein after Ni-NTA chromatography. Protein was assayed in the presence of 5 mM CHAPS with 1 mM ATP and 12 mM metal (Mg or Mn) as indicated. Values represent the means for duplicate determinations of a representative assay.

Figure 4. Biochemical characterization of the Rv1647 catalytic domain protein

(A) Purified protein was assayed at indicated concentrations with 1 mM MnATP and 10 mM free Mn, and initial reaction rates are plotted. Results are representative of assays performed twice in duplicate and means±S.E.M. are shown. (B) Rv1647 protein (∼170 nM) was assayed in the presence of different concentrations of substrate (MnATP) as indicated in the presence of a fixed amount of 10 mM free Mn. Assays were performed three times in duplicate and means±S.E.M. (n=6) are shown. (C) Protein (∼170 nM) was assayed in the presence of a fixed concentration of 1 mM MnATP and different concentrations of free Mn as indicated. Experiments were performed twice in duplicate and means±S.E.M. (n=4) are shown.

Figure 5. Oligomeric status of the catalytic domain of Rv1647

Cross-linking was performed using 2 mM DSS or DSG or in the presence of solvent alone (DMSO), and in the presence or absence of 1 mM ATP and 12 mM Mn as indicated. Samples were transferred on to PVDF and analysed by Western blotting using an affinity-purified antibody against the Rv1647 catalytic domain.

Figure 6. Mutational studies on the catalytic domain of Rv1647

(A) The adenylate cyclase activity of indicated proteins (∼690 nM) when assayed with 1 mM MnATP and 10 mM free Mn is shown. Results shown represent means±S.E.M. (n=4). Coomassie-stained gel showing the purified mutant Rv1647 catalytic domain proteins is shown as an inset. (B) Schematic representation of complementation between C1- and C2-like Rv1647 proteins. An active site with the critical functional residues from both protomers will be competent catalytically, whereas other combinations (C1–C1 or C2–C2 homodimers) will not. (C) The activities of Rv1647_D147A alone (∼70 nM; ◇) and in the presence of different concentrations of Rv1625c_K296E/D365C (●) are shown. Activities of Rv1647_K187E/D241C at different concentrations in the absence (□) or presence of approx. 70 nM of Rv1647_D147A protein (■) are shown. Assays were performed twice and means±S.E.M. are shown (n=4).

Figure 7. Effects of tyrphostin A25 on Rv1647 and Rv1625c

Proteins were assayed in the presence of indicated concentrations of tyrphostin A25 in 1% DMSO as solvent. Values are expressed as percentage activity observed in the presence of a similar amount of solvent alone. These activities were approx. 3.5 μmol of cAMP produced·min⁻¹·(mg of protein)⁻¹ for Rv1647 and approx. 300 nmol of cAMP produced·min⁻¹·(mg of protein)⁻¹ for Rv1625c. Values are representative of assays performed at least three times and means±S.E.M. are shown (n=6).

Figure 8. Expression and activity of the ML1399 protein

Coomassie-stained SDS/polyacrylamide gel showing the ML1399 catalytic domain protein of molecular mass approx. 29 kDa purified using Ni-NTA Sepharose. Western-blot analysis of the purified protein was performed with affinity-purified antibody to Rv1647. Adenylate cyclase activity of the protein (∼680 nM) was performed using 1 mM ATP and 12 mM Mn at pH 8.5. Results shown represent the means±S.E.M. for assays performed twice in duplicates (n=4).

Figure 9. Expression of Rv1647 and Rv1625c in M. tuberculosis and ML1399 in M. leprae

(A) Whole-cell lysates and subcellular fractions (20 μg of protein) of the M. tuberculosis H37Rv strain (M.tubH37Rv) and M. leprae were obtained from Colorado State University and subjected to Western-blot analysis using an affinity-purified antibody raised against the Rv1647 catalytic domain. (B) Lysates were used for Western-blot analysis using the affinity-purified antibody against Rv1625c. The same fractions were also probed using a mAb (HBT-12 at 1:100 dilution) against a phosphate transporter (PstS-1). W, whole-cell lysates; Cyt, cytosol; M, plasma membrane; CW, cell wall; CFP, culture filtrate proteins. Proteins were visualized using enhanced chemiluminescence.