Comparison of AC and GC activity of mutants with the wild type at pH 7.5 and 9.0. Mean ± SEM are shown from experiments performed twice with quadruplicates.

<p>– <i>sign in the last two columns indicates percentage decrease in GC activity relative to the wild type.</i></p><p>Comparison of AC and GC activity of mutants with the wild type at pH 7.5 and 9.0. Mean ± SEM are shown from experiments performed twice with quadruplicates.</p

Amino acid sequence alignment of the catalytic region of adenylyl and guanylylcyclases using T-COFFEE web server.

<p>First column has the GI accession numbers of proteins available in National Center for Biotechnology Information database followed by type of nucleotidyl cyclase and species names (M.ava: <i>Mycobacterium avium</i>; C. fam: <i>Canis lupus familiaris</i>; R. nor: <i>Rattusnorvegicus</i>; Nostoc: <i>Nostoc sp. PCC 7120</i>; D.mel: <i>Drosophila melanogaster</i>; Synecho: <i>Synechocystis sp. PCC 6803</i>; P. tet: <i>Paramecium tetraurelia</i>; M.sec:<i>Manducasexta</i>; H.sap: <i>Homo sapiens</i>; A.var: <i>Anabaena variabilis ATCC 29413</i>; D. dis: <i>Dictyosteliumdiscoideum</i>; C. rei: Chlamydomonasreinhardtii). The second column indicates the amino acid position of the domain in the respective sequences. Critical metal binding residues are indicated by•, substrate specifying residues by and transition state stabilizing residues are depicted by▪.</p

AC and GC assays of Ma1120 and mutants were conducted at varied concentrations of ATP and GTP ( 0 to 2000 µM) and their k_cat, K_m and k_cat/K_m that were calculated using GraphPad prism software are shown.

<p>All assays were performed at pH 8.</p><p>AC and GC assays of Ma1120 and mutants were conducted at varied concentrations of ATP and GTP ( 0 to 2000 µM) and their k_cat, K_m and k_cat/K_m that were calculated using GraphPad prism software are shown.</p

Mutational Analysis Gives Insight into Substrate Preferences of a Nucleotidyl Cyclase from <i>Mycobacterium avium</i>

<div><p>Mutational, crystallographic and phylogenetic analysis of nucleotidyl cyclases have been used to understand how these enzymes discriminate between substrates. Ma1120, a class III adenylyl cyclase (AC) from <i>Mycobacterium avium</i>, was used as a model to study the amino acid residues that determine substrate preference, by systematically replacing ATP specifying residues with those known to specify GTP. This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH. Replacement of key residues lysine (101) and aspartate (157) with residues conserved across GCs by site directed mutagenesis, led to a marked improvement in GC activity and a decrease in AC activity. This could be correlated to the presence and strength of the hydrogen bond between the second substrate binding residue (157) and the base of the nucleotide triphosphate. This is substantiated by the fact that the pH optimum is highly dependent on the amino acid residues present at positions 101 and 157.</p></div

Sequence of the primers along with their respective T_m values.

<p>Sequence of the primers along with their respective T_m values.</p

Variation of adenylyl cyclase and guanylyl cyclase activity of Ma1120, K101E, D157C and K101E/D157C with pH.Adenylyl and guanylyl cyclase assays of Ma1120 andits mutants were performed at different pH (5, 6, 7, 7.5, 8, 9, 10 and 11) conditions using triple buffer (MES, HEPES and diethanolamine) at 50 mM concentration and enzyme concentration of 500 nM.

<p>cAMP and cGMP measurements were carried out by radioimmunoassay.Mean ±SEM are shown from experiments performed twice with quadruplicates.</p

Substrate specificity determinants of class III nucleotidyl cyclases

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. Database Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120(CHD)+ATP.Ca2+), 5D0E (Ma1120(CHD)+GTP.Ca2+), 5D0H (Ma1120(CHD)(KDA -> EGY)+ATP.Ca2+), 5D0G (Ma1120(CHD)(KDA -> EGY)+GTP.Ca2+). Enzymes Adenylyl cyclase (EC number: 4.6.1.1)

Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase

An adenylyl cyclase from Mycobacterium avium, Mal 120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Mal 120 in a monomeric form and its truncated construct as a dimer. Mal 120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional alpha-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. (C) 2015 Elsevier Inc. All rights reserved