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.

Substrate specificity determinants of class III nucleotidyl cyclases

“…A). Mutation of Ala216 to Asn216, a conserved residue in cyclases, leads to a drastic reduction in AC activity in Ma1120 . This is a consequence of steric clashes between the ligand and the side chain of Asn at this position.…”

“…A double mutant of Ma1120‐∆N52 in which the adenine‐specifying residues Lys153 and Asp209 were mutated to guanine‐specifying Glu and Gly residues showed enhanced GC activity . In Cya2, a tyrosine residue in the binding pocket, Tyr572, was predicted to aid in GTP stabilization by making a hydrogen bond with guanine .…”

“…The corresponding residue in Ma1120, Ala219, was also mutated to tyrosine to generate a triple mutant (Ma1120 CHD (KDA→EGY)), to enhance the affinity for GTP. Biochemical characterization of the triple mutant showed that it exhibits equal specificity ( k cat / K m = 0.22) for both the substrates . To understand the exact mode of binding of the substrates to this enzyme, we crystallized Ma1120 CHD (KDA→EGY) in the presence of ATP/GTP and Ca 2+ .…”

“…One of the 12 ACs identified in Mycobacterium avium is Ma1120 which has 275 residues. Its deletion construct, Ma1120‐∆N52, was shown to possess residual GC activity which increased when its ATP‐specifying residues were mutated to GTP‐specifying residues of GCs . Ma1120‐∆N52 has 2% residual GC activity compared to its AC activity.…”

“…In an attempt to convert it to a GC, a triple mutant was generated by mutating two adenine‐specifying residues to guanine‐specifying residues (K153→E and D209→G); and an alanine to tyrosine (A219→Y) to enhance GTP binding as observed in Cya2 . This mutant shows about fourfold increase in GC activity and a threefold decrease in AC activity (Table ). Previously, we reported the structures of Ma1120‐∆N52 in a monomeric form (residues 53–275) and the complexes of the active dimeric form of the cyclase homology domain of the enzyme (Ma1120 CHD : residues 106–275) with 2′,5′‐dideoxy‐3′‐ATP and pyrophosphate along with metal ions .…”