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Mutational analysis gives insight into substrate preferences of a nucleotidyl cyclase from Mycobacterium avium.

Syed W, Colaςo M, Misquith S - PLoS ONE (2014)

Bottom Line: This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, St. Joseph's College, Bangalore, India.

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.

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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.cAMP and cGMP measurements were carried out by radioimmunoassay.Mean ±SEM are shown from experiments performed twice with quadruplicates.
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pone-0109358-g003: 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.cAMP and cGMP measurements were carried out by radioimmunoassay.Mean ±SEM are shown from experiments performed twice with quadruplicates.

Mentions: AC and GC activity of Ma1120 and some of its mutants was carried out at pH varying from 5 to 11. While the optimum pH of Ma1120 was 7.5 when ATP was the substrate, it was 9.0 for GTP as seen in Fig. 3. For the mutants K101E, D157C and K101E/D157C the pH optimum was found to be pH 9 irrespective of whether the substrate was ATP or GTP. For the other mutants the AC and GC activity was determined at pH 7.5 and 9.0. In all cases the activity was higher at pH 9.0 compared to pH 7.5 (Fig. S2). Thus the activities of the mutants were compared with respect to the original activity possessed by the wild type at pH 7.5 and pH 9.0 (Table 2). Interestingly, the AC activity of the single mutant K101E which was practically abolished at pH 7.5 retained at least 40% of its AC activity at pH 9.0. However GC activity was enhanced by about four-fold at pH 7.5 while there was an 80% increase at pH 9.0.


Mutational analysis gives insight into substrate preferences of a nucleotidyl cyclase from Mycobacterium avium.

Syed W, Colaςo M, Misquith S - PLoS ONE (2014)

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.cAMP and cGMP measurements were carried out by radioimmunoassay.Mean ±SEM are shown from experiments performed twice with quadruplicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215837&req=5

pone-0109358-g003: 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.cAMP and cGMP measurements were carried out by radioimmunoassay.Mean ±SEM are shown from experiments performed twice with quadruplicates.
Mentions: AC and GC activity of Ma1120 and some of its mutants was carried out at pH varying from 5 to 11. While the optimum pH of Ma1120 was 7.5 when ATP was the substrate, it was 9.0 for GTP as seen in Fig. 3. For the mutants K101E, D157C and K101E/D157C the pH optimum was found to be pH 9 irrespective of whether the substrate was ATP or GTP. For the other mutants the AC and GC activity was determined at pH 7.5 and 9.0. In all cases the activity was higher at pH 9.0 compared to pH 7.5 (Fig. S2). Thus the activities of the mutants were compared with respect to the original activity possessed by the wild type at pH 7.5 and pH 9.0 (Table 2). Interestingly, the AC activity of the single mutant K101E which was practically abolished at pH 7.5 retained at least 40% of its AC activity at pH 9.0. However GC activity was enhanced by about four-fold at pH 7.5 while there was an 80% increase at pH 9.0.

Bottom Line: This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, St. Joseph's College, Bangalore, India.

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.

Show MeSH
Related in: MedlinePlus