Limits...
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.

Show MeSH

Related in: MedlinePlus

Amino acid sequence alignment of the catalytic region of adenylyl and guanylylcyclases using T-COFFEE web server.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: Mycobacterium avium; C. fam: Canis lupus familiaris; R. nor: Rattusnorvegicus; Nostoc: Nostoc sp. PCC 7120; D.mel: Drosophila melanogaster; Synecho: Synechocystis sp. PCC 6803; P. tet: Paramecium tetraurelia; M.sec:Manducasexta; H.sap: Homo sapiens; A.var: Anabaena variabilis ATCC 29413; D. dis: Dictyosteliumdiscoideum; 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▪.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215837&req=5

pone-0109358-g001: Amino acid sequence alignment of the catalytic region of adenylyl and guanylylcyclases using T-COFFEE web server.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: Mycobacterium avium; C. fam: Canis lupus familiaris; R. nor: Rattusnorvegicus; Nostoc: Nostoc sp. PCC 7120; D.mel: Drosophila melanogaster; Synecho: Synechocystis sp. PCC 6803; P. tet: Paramecium tetraurelia; M.sec:Manducasexta; H.sap: Homo sapiens; A.var: Anabaena variabilis ATCC 29413; D. dis: Dictyosteliumdiscoideum; 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▪.

Mentions: Multiple sequence alignment of Ma1120 cyclase domain with representative cyclase domains of ACs and GCs (Fig. 1) shows that the substrate binding residues, lysine (K) and aspartate (D) are conserved in ACs across species. In GCs, glutamate is present instead of K while in place of aspartate, one observes a variety of seemingly unrelated amino acid residues that include cysteine(C), serine(S), threonine(T), histidine(H), alanine(A) or glycine(G).


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

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

Amino acid sequence alignment of the catalytic region of adenylyl and guanylylcyclases using T-COFFEE web server.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: Mycobacterium avium; C. fam: Canis lupus familiaris; R. nor: Rattusnorvegicus; Nostoc: Nostoc sp. PCC 7120; D.mel: Drosophila melanogaster; Synecho: Synechocystis sp. PCC 6803; P. tet: Paramecium tetraurelia; M.sec:Manducasexta; H.sap: Homo sapiens; A.var: Anabaena variabilis ATCC 29413; D. dis: Dictyosteliumdiscoideum; 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▪.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109358-g001: Amino acid sequence alignment of the catalytic region of adenylyl and guanylylcyclases using T-COFFEE web server.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: Mycobacterium avium; C. fam: Canis lupus familiaris; R. nor: Rattusnorvegicus; Nostoc: Nostoc sp. PCC 7120; D.mel: Drosophila melanogaster; Synecho: Synechocystis sp. PCC 6803; P. tet: Paramecium tetraurelia; M.sec:Manducasexta; H.sap: Homo sapiens; A.var: Anabaena variabilis ATCC 29413; D. dis: Dictyosteliumdiscoideum; 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▪.
Mentions: Multiple sequence alignment of Ma1120 cyclase domain with representative cyclase domains of ACs and GCs (Fig. 1) shows that the substrate binding residues, lysine (K) and aspartate (D) are conserved in ACs across species. In GCs, glutamate is present instead of K while in place of aspartate, one observes a variety of seemingly unrelated amino acid residues that include cysteine(C), serine(S), threonine(T), histidine(H), alanine(A) or glycine(G).

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