title: Mutational analysis gives insight into substrate preferences of a nucleotidyl cyclase from Mycobacterium avium.
abstract: 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 Mycobacterium avium, 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.
authors.affiliation: Department of Chemistry, St. Joseph's College, Bangalore, India.
authors.forename: Wajeed
authors.initials: W
authors.lastname: Syed
authors.affiliation: Department of Chemistry, St. Joseph's College, Bangalore, India.
authors.forename: Melwin
authors.initials: M
authors.lastname: Colaςo
authors.affiliation: Department of Chemistry, St. Joseph's College, Bangalore, India.
authors.forename: Sandra
authors.initials: S
authors.lastname: Misquith
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009162:Mycobacterium avium / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; C421784:adenylate cyclase 3