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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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Coomassie stained 15% SDS- polyacrylamide gel showing purified Ma1120 and representative Ma1120 mutant proteins.M: Marker, P: pellet, S: supernatant, FT: flowthrough, WT: Ma1120, KE: K101E, DC: D157C and KEDC: K101E/D157C.
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pone-0109358-g002: Coomassie stained 15% SDS- polyacrylamide gel showing purified Ma1120 and representative Ma1120 mutant proteins.M: Marker, P: pellet, S: supernatant, FT: flowthrough, WT: Ma1120, KE: K101E, DC: D157C and KEDC: K101E/D157C.

Mentions: Ma1120 and the mutant proteins were expressed in E.coli BL21DE3 cells and purified using Ni-NTA agarose. The proteins were found to be pure by SDS-PAGE and banded at about 29 kDa. In Fig. 2 the SDS-PAGE profile of representative proteins has been shown and the rest are given in Fig. S1.


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

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

Coomassie stained 15% SDS- polyacrylamide gel showing purified Ma1120 and representative Ma1120 mutant proteins.M: Marker, P: pellet, S: supernatant, FT: flowthrough, WT: Ma1120, KE: K101E, DC: D157C and KEDC: K101E/D157C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109358-g002: Coomassie stained 15% SDS- polyacrylamide gel showing purified Ma1120 and representative Ma1120 mutant proteins.M: Marker, P: pellet, S: supernatant, FT: flowthrough, WT: Ma1120, KE: K101E, DC: D157C and KEDC: K101E/D157C.
Mentions: Ma1120 and the mutant proteins were expressed in E.coli BL21DE3 cells and purified using Ni-NTA agarose. The proteins were found to be pure by SDS-PAGE and banded at about 29 kDa. In Fig. 2 the SDS-PAGE profile of representative proteins has been shown and the rest are given in Fig. S1.

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