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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 coevolutionary analysis.(A) A matrix of rZNMI scores by residue for the PMM/PGM MSA (lower right triangle). Range (0–100) indicates the reproducibility of the residue couplings from the data resampling (Methods). Blue dashed lines separate regions of the matrix according to the domain of the protein. (B) Plots showing the degree (summation of reproducible couplings), entropy and gaps for each column of the rZNMI matrix. Colored boxes at top indicate the four domains of PMM/PGM: domain 1 (residues 1–154), domain 2 (residues 154–256), domain 3 (residues 257–368), and domain 4 (residues 369–463); numbers according to the P. aeruginosa enzyme sequence.
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pone-0038114-g001: Results of the coevolutionary analysis.(A) A matrix of rZNMI scores by residue for the PMM/PGM MSA (lower right triangle). Range (0–100) indicates the reproducibility of the residue couplings from the data resampling (Methods). Blue dashed lines separate regions of the matrix according to the domain of the protein. (B) Plots showing the degree (summation of reproducible couplings), entropy and gaps for each column of the rZNMI matrix. Colored boxes at top indicate the four domains of PMM/PGM: domain 1 (residues 1–154), domain 2 (residues 154–256), domain 3 (residues 257–368), and domain 4 (residues 369–463); numbers according to the P. aeruginosa enzyme sequence.

Mentions: A matrix of the resampled ZNMI (rZNMI) scores for all possible residue position pairs in the PMM/PGM MSA is given in Fig. 1A. Also, for comparison, Fig.1B shows per residue plots of the degree (summation of reproducible couplings), sequence entropy, and gaps for the MSA. The matrix in Fig. 1A is notably sparse due to application of the 100% reproducibility criterion. Despite this, the number of high-scoring residue pairs from the rZNMI analysis is still quite large (157 couplings over 74 sequence positions), and difficult to assess on an individual basis. In general, however, it can be seen that high-scoring couplings are scattered throughout the sequence, although with higher density in the C-terminal half of the protein. When considered according to specific domains, a propensity of high-scoring residue pairs occur within domain 3 (intradomain couplings) and between residues from domains 3 and 4 of the protein (interdomain couplings). (For reference, a structural overview of the domain architecture of PMM/PGM is given on Fig. S1). As expected, Fig. 1B shows that residues with high degrees of coupling do not coincide with regions of low sequence entropy (high conservation). It also shows that our MSA does not have extended continuous regions of high degree, which have been correlated with sequence misalignments [32].


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 coevolutionary analysis.(A) A matrix of rZNMI scores by residue for the PMM/PGM MSA (lower right triangle). Range (0–100) indicates the reproducibility of the residue couplings from the data resampling (Methods). Blue dashed lines separate regions of the matrix according to the domain of the protein. (B) Plots showing the degree (summation of reproducible couplings), entropy and gaps for each column of the rZNMI matrix. Colored boxes at top indicate the four domains of PMM/PGM: domain 1 (residues 1–154), domain 2 (residues 154–256), domain 3 (residues 257–368), and domain 4 (residues 369–463); numbers according to the P. aeruginosa enzyme sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038114-g001: Results of the coevolutionary analysis.(A) A matrix of rZNMI scores by residue for the PMM/PGM MSA (lower right triangle). Range (0–100) indicates the reproducibility of the residue couplings from the data resampling (Methods). Blue dashed lines separate regions of the matrix according to the domain of the protein. (B) Plots showing the degree (summation of reproducible couplings), entropy and gaps for each column of the rZNMI matrix. Colored boxes at top indicate the four domains of PMM/PGM: domain 1 (residues 1–154), domain 2 (residues 154–256), domain 3 (residues 257–368), and domain 4 (residues 369–463); numbers according to the P. aeruginosa enzyme sequence.
Mentions: A matrix of the resampled ZNMI (rZNMI) scores for all possible residue position pairs in the PMM/PGM MSA is given in Fig. 1A. Also, for comparison, Fig.1B shows per residue plots of the degree (summation of reproducible couplings), sequence entropy, and gaps for the MSA. The matrix in Fig. 1A is notably sparse due to application of the 100% reproducibility criterion. Despite this, the number of high-scoring residue pairs from the rZNMI analysis is still quite large (157 couplings over 74 sequence positions), and difficult to assess on an individual basis. In general, however, it can be seen that high-scoring couplings are scattered throughout the sequence, although with higher density in the C-terminal half of the protein. When considered according to specific domains, a propensity of high-scoring residue pairs occur within domain 3 (intradomain couplings) and between residues from domains 3 and 4 of the protein (interdomain couplings). (For reference, a structural overview of the domain architecture of PMM/PGM is given on Fig. S1). As expected, Fig. 1B shows that residues with high degrees of coupling do not coincide with regions of low sequence entropy (high conservation). It also shows that our MSA does not have extended continuous regions of high degree, which have been correlated with sequence misalignments [32].

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