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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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Structural networks in the domain 4 interface.(A) Schematic of the domain 4 interface of P. aeruginosa PMM/PGM, highlighting the residues involved and types of interactions. Hydrogen bonds are shown as dashed lines; van der Waals contacts as dotted lines. Yellow line approximates location of the inter-domain interface. Residues in the top cliques are highlighted with blue shading; residues selected for mutation (or previously mutated) are outlined in purple. Residues where contact involves backbone atoms are indicated with “bb” in the residue label; backbone connections between sequential residues are shown with a solid gray line. Non-interface residues are shown in gray font. Interactions represent a compilation of those seen in various crystal structures of PMM/PGM; not all interactions shown are found in each structure. See Table S2 for a full listing of interactions of clique residues. (B) A close-up of the domain 4 interface on the structure of PMM/PGM. Residues in the interface that are not top clique residues are shown in yellow; other colors as in panel A. Labels of residues selected for mutation are highlighted in purple boxes.
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pone-0038114-g004: Structural networks in the domain 4 interface.(A) Schematic of the domain 4 interface of P. aeruginosa PMM/PGM, highlighting the residues involved and types of interactions. Hydrogen bonds are shown as dashed lines; van der Waals contacts as dotted lines. Yellow line approximates location of the inter-domain interface. Residues in the top cliques are highlighted with blue shading; residues selected for mutation (or previously mutated) are outlined in purple. Residues where contact involves backbone atoms are indicated with “bb” in the residue label; backbone connections between sequential residues are shown with a solid gray line. Non-interface residues are shown in gray font. Interactions represent a compilation of those seen in various crystal structures of PMM/PGM; not all interactions shown are found in each structure. See Table S2 for a full listing of interactions of clique residues. (B) A close-up of the domain 4 interface on the structure of PMM/PGM. Residues in the interface that are not top clique residues are shown in yellow; other colors as in panel A. Labels of residues selected for mutation are highlighted in purple boxes.

Mentions: Four of the top clique residues meet the two criteria described above: D261, K285, R410, and R432. Their structural roles and interactions in the domain 4 interface are summarized in Fig. 4. This figure is a compilation of observed interactions as these vary depending on enzyme conformer and identity of bound ligand (see Table S2 for a full listing). D261 makes contacts with other residues in domain 3, and also across the domain interface with R432 in several of the enzyme-ligand complexes. K285 makes multiple contacts across the interface with residues in domain 4, including the backbone of R432. However, it is located on the periphery of the active site, and, as noted previously, also participates in ligand contacts. Hence, this residue acts as a bridge between the active site and the domain 4 interface, and is unique in this regard from the others. R410 contacts backbone atoms of residues 284 and 286, which flank K285. Most bond interactions between the top clique residues are between atoms in the side chain of one and the backbone of another. The only direct side chain-side chain interaction between two top clique residues is for D261 and R432.


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)

Structural networks in the domain 4 interface.(A) Schematic of the domain 4 interface of P. aeruginosa PMM/PGM, highlighting the residues involved and types of interactions. Hydrogen bonds are shown as dashed lines; van der Waals contacts as dotted lines. Yellow line approximates location of the inter-domain interface. Residues in the top cliques are highlighted with blue shading; residues selected for mutation (or previously mutated) are outlined in purple. Residues where contact involves backbone atoms are indicated with “bb” in the residue label; backbone connections between sequential residues are shown with a solid gray line. Non-interface residues are shown in gray font. Interactions represent a compilation of those seen in various crystal structures of PMM/PGM; not all interactions shown are found in each structure. See Table S2 for a full listing of interactions of clique residues. (B) A close-up of the domain 4 interface on the structure of PMM/PGM. Residues in the interface that are not top clique residues are shown in yellow; other colors as in panel A. Labels of residues selected for mutation are highlighted in purple boxes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369874&req=5

pone-0038114-g004: Structural networks in the domain 4 interface.(A) Schematic of the domain 4 interface of P. aeruginosa PMM/PGM, highlighting the residues involved and types of interactions. Hydrogen bonds are shown as dashed lines; van der Waals contacts as dotted lines. Yellow line approximates location of the inter-domain interface. Residues in the top cliques are highlighted with blue shading; residues selected for mutation (or previously mutated) are outlined in purple. Residues where contact involves backbone atoms are indicated with “bb” in the residue label; backbone connections between sequential residues are shown with a solid gray line. Non-interface residues are shown in gray font. Interactions represent a compilation of those seen in various crystal structures of PMM/PGM; not all interactions shown are found in each structure. See Table S2 for a full listing of interactions of clique residues. (B) A close-up of the domain 4 interface on the structure of PMM/PGM. Residues in the interface that are not top clique residues are shown in yellow; other colors as in panel A. Labels of residues selected for mutation are highlighted in purple boxes.
Mentions: Four of the top clique residues meet the two criteria described above: D261, K285, R410, and R432. Their structural roles and interactions in the domain 4 interface are summarized in Fig. 4. This figure is a compilation of observed interactions as these vary depending on enzyme conformer and identity of bound ligand (see Table S2 for a full listing). D261 makes contacts with other residues in domain 3, and also across the domain interface with R432 in several of the enzyme-ligand complexes. K285 makes multiple contacts across the interface with residues in domain 4, including the backbone of R432. However, it is located on the periphery of the active site, and, as noted previously, also participates in ligand contacts. Hence, this residue acts as a bridge between the active site and the domain 4 interface, and is unique in this regard from the others. R410 contacts backbone atoms of residues 284 and 286, which flank K285. Most bond interactions between the top clique residues are between atoms in the side chain of one and the backbone of another. The only direct side chain-side chain interaction between two top clique residues is for D261 and R432.

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