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A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

Lee Y, Mick J, Furdui C, Beamer LJ - PLoS ONE (2012)

Bottom Line: For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme.The results of these studies are interpreted in the context of structural and functional data on PMM/PGM.Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, Columbia, Missouri, United States of America.

ABSTRACT
Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

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Results of the clique analysis.(A) Graph illustrating the connections between the 12 residues (ovals) in the top cliques of PMM/PGM (see text). Gray shading highlights residues characterized in mutagenesis studies. Lines connecting ovals indicate that two residues are neighbors (in the same clique). (B) Upper right triangle: A contact map showing the distance between the closest atoms for each pair of top clique residues, from 0 (blue) to 10 Å (red); see color bar. Residues belonging to domains 3 and 4 of the protein are highlighted by brackets on right. The physical proximity of top clique residues in the domain 4 interface can be easily visualized by the patches of blue/green. Lower left triangle. An array of bi-variate histograms showing the joint amino acid identities between the top clique residues. Axes for the array are residue numbers; each histogram is labeled with amino acid types along axes i and j. Blue indicates low joint residue identity (0.0); red indicates high (1.0). Joint identities that occur at a frequency of less than 5% were removed for simplicity. Each histogram is normalized to its sum, and since all residues shown are co-varying (i.e., not completely conserved), the maximal score (red) is not possible for any pair. Residues along the bottom axis highlighted by asterisks were subject to study by mutagenesis.
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pone-0038114-g002: Results of the clique analysis.(A) Graph illustrating the connections between the 12 residues (ovals) in the top cliques of PMM/PGM (see text). Gray shading highlights residues characterized in mutagenesis studies. Lines connecting ovals indicate that two residues are neighbors (in the same clique). (B) Upper right triangle: A contact map showing the distance between the closest atoms for each pair of top clique residues, from 0 (blue) to 10 Å (red); see color bar. Residues belonging to domains 3 and 4 of the protein are highlighted by brackets on right. The physical proximity of top clique residues in the domain 4 interface can be easily visualized by the patches of blue/green. Lower left triangle. An array of bi-variate histograms showing the joint amino acid identities between the top clique residues. Axes for the array are residue numbers; each histogram is labeled with amino acid types along axes i and j. Blue indicates low joint residue identity (0.0); red indicates high (1.0). Joint identities that occur at a frequency of less than 5% were removed for simplicity. Each histogram is normalized to its sum, and since all residues shown are co-varying (i.e., not completely conserved), the maximal score (red) is not possible for any pair. Residues along the bottom axis highlighted by asterisks were subject to study by mutagenesis.

Mentions: The union of the five largest cliques (hereafter called the “top cliques”) results in a set of just 12 residue positions of PMM/PGM, as many of the residues are found in more than one clique (Table 1). The network of relationships between the residues in the top cliques is illustrated in Fig. 2A, where each line connecting a pair of residues indicates that they are neighbors in a clique. Residue numbers on this figure and throughout manuscript refer to P. aeruginosa PMM/PGM. The connectedness of these residue positions varies considerably: some belong to only one clique (one set of neighbors), while other residues belong to multiple cliques and thus have many more neighbors. We note that each of the top cliques contains at least one residue from domain 3 of the protein and one from domain 4 (Table 1).


A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

Lee Y, Mick J, Furdui C, Beamer LJ - PLoS ONE (2012)

Results of the clique analysis.(A) Graph illustrating the connections between the 12 residues (ovals) in the top cliques of PMM/PGM (see text). Gray shading highlights residues characterized in mutagenesis studies. Lines connecting ovals indicate that two residues are neighbors (in the same clique). (B) Upper right triangle: A contact map showing the distance between the closest atoms for each pair of top clique residues, from 0 (blue) to 10 Å (red); see color bar. Residues belonging to domains 3 and 4 of the protein are highlighted by brackets on right. The physical proximity of top clique residues in the domain 4 interface can be easily visualized by the patches of blue/green. Lower left triangle. An array of bi-variate histograms showing the joint amino acid identities between the top clique residues. Axes for the array are residue numbers; each histogram is labeled with amino acid types along axes i and j. Blue indicates low joint residue identity (0.0); red indicates high (1.0). Joint identities that occur at a frequency of less than 5% were removed for simplicity. Each histogram is normalized to its sum, and since all residues shown are co-varying (i.e., not completely conserved), the maximal score (red) is not possible for any pair. Residues along the bottom axis highlighted by asterisks were subject to study by mutagenesis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369874&req=5

pone-0038114-g002: Results of the clique analysis.(A) Graph illustrating the connections between the 12 residues (ovals) in the top cliques of PMM/PGM (see text). Gray shading highlights residues characterized in mutagenesis studies. Lines connecting ovals indicate that two residues are neighbors (in the same clique). (B) Upper right triangle: A contact map showing the distance between the closest atoms for each pair of top clique residues, from 0 (blue) to 10 Å (red); see color bar. Residues belonging to domains 3 and 4 of the protein are highlighted by brackets on right. The physical proximity of top clique residues in the domain 4 interface can be easily visualized by the patches of blue/green. Lower left triangle. An array of bi-variate histograms showing the joint amino acid identities between the top clique residues. Axes for the array are residue numbers; each histogram is labeled with amino acid types along axes i and j. Blue indicates low joint residue identity (0.0); red indicates high (1.0). Joint identities that occur at a frequency of less than 5% were removed for simplicity. Each histogram is normalized to its sum, and since all residues shown are co-varying (i.e., not completely conserved), the maximal score (red) is not possible for any pair. Residues along the bottom axis highlighted by asterisks were subject to study by mutagenesis.
Mentions: The union of the five largest cliques (hereafter called the “top cliques”) results in a set of just 12 residue positions of PMM/PGM, as many of the residues are found in more than one clique (Table 1). The network of relationships between the residues in the top cliques is illustrated in Fig. 2A, where each line connecting a pair of residues indicates that they are neighbors in a clique. Residue numbers on this figure and throughout manuscript refer to P. aeruginosa PMM/PGM. The connectedness of these residue positions varies considerably: some belong to only one clique (one set of neighbors), while other residues belong to multiple cliques and thus have many more neighbors. We note that each of the top cliques contains at least one residue from domain 3 of the protein and one from domain 4 (Table 1).

Bottom Line: For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme.The results of these studies are interpreted in the context of structural and functional data on PMM/PGM.Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Missouri, Columbia, Missouri, United States of America.

ABSTRACT
Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

Show MeSH
Related in: MedlinePlus