[Show abstract][Hide abstract] ABSTRACT: The α-d-phosphohexomutase superfamily comprises enzymes involved in carbohydrate metabolism that are found in all kingdoms of life. Recent biophysical studies have shown for the first time that several of these enzymes exist as dimers in solution, prompting an examination of the oligomeric state of all proteins of known structure in the superfamily (11 different proteins; 31 crystal structures) via computational and experimental analyses. We find that these proteins range in quaternary structure from monomers to tetramers, with 6 of the 11 known structures being likely oligomers. The oligomeric state of these proteins not only is associated in some cases with enzyme subgroup (i.e., substrate specificity) but also appears to depend on domain of life, with the two archaeal proteins existing as higher-order oligomers. Within the oligomers, three distinct interfaces are observed, one of which is found in both archaeal and bacterial proteins. Normal mode analysis shows that the topological arrangement of the oligomers permits domain 4 of each protomer to move independently as required for catalysis. Our analysis suggests that the advantages associated with protein flexibility in this enzyme family are of sufficient importance to be maintained during the evolution of multiple independent oligomers. This study is one of the first showing that global motions may be conserved not only within protein families but also across members of a superfamily with varying oligomeric structures.
[Show abstract][Hide abstract] 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.
PLoS ONE 06/2012; 7(6):e38114. DOI:10.1371/journal.pone.0038114 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.
[Show abstract][Hide abstract] ABSTRACT: Phosphoglucosamine mutase (PNGM) is a bacterial enzyme that participates in the peptidoglycan biosynthetic pathway. Recent crystal structures of PNGM from two bacterial pathogens, Bacillus anthracis and Francisella tularensis, have revealed key structural features of this enzyme for the first time. Here, we follow up on several novel findings from the crystallographic studies, including the observation of a structurally conserved interface between polypeptide chains and conformational variability of the C-terminal domain. Small-angle X-ray scattering of B. anthracis PNGM shows that this protein is a dimer in solution. Comparisons of the four independent polypeptide chains from the two structures reveals conserved residues and structural changes involved in the conformational variability, as well as a significant rotation of the C-terminal domain, of nearly 60°, between the most divergent conformers. Furthermore, the fluctuation dynamics of PNGM are examined via normal mode analyses. The most mobile region of the protein is its C-terminal domain, consistent with observations from the crystal structures. Large regions of correlated, collective motions are identified exclusively for the dimeric state of the protein, comprising both contiguous and noncontiguous structural domains. The motions observed in the lowest frequency normal mode of the dimer result in dynamically coupled opening and closing of the two active sites. The global motions identified in this study support the importance of the conformational change of PNGM in function, and suggest that the dimeric state of this protein may confer advantages consistent with its evolutionary conservation.
Structured digital abstract
[Show abstract][Hide abstract] ABSTRACT: Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme that participates in the cytoplasmic steps
of peptidoglycan biosynthesis. As peptidoglycan is essential for bacterial survival and is absent in humans, enzymes in this
pathway have been the focus of intensive inhibitor design efforts. Many aspects of the structural biology of the peptidoglycan
pathway have been elucidated, with the exception of the PNGM structure. We present here the crystal structure of PNGM from
the human pathogen and bioterrorism agent Bacillus anthracis. The structure reveals key residues in the large active site cleft of the enzyme which likely have roles in catalysis and
specificity. A large conformational change of the C-terminal domain of PNGM is observed when comparing two independent molecules
in the crystal, shedding light on both the apo- and ligand-bound conformers of the enzyme. Crystal packing analyses and dynamic
light scattering studies suggest that the enzyme is a dimer in solution. Multiple sequence alignments show that residues in
the dimer interface are conserved, suggesting that many PNGM enzymes adopt this oligomeric state. This work lays the foundation
for the development of inhibitors for PNGM enzymes from human pathogens.
Journal of bacteriology 06/2011; 193(16):4081-7. DOI:10.1128/JB.00418-11 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the enzyme phosphoglucomutase from Salmonella typhimurium (StPGM) is reported at 1.7 A resolution. This is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity. A comparison of the active site of StPGM with that of other phosphoglucomutases reveals conserved residues that are likely involved in catalysis and ligand binding for the entire enzyme family. An alternate crystal form of StPGM and normal mode analysis give insights into conformational changes of the C-terminal domain that occur upon ligand binding. A novel observation from the StPGM structure is an apparent dimer in the asymmetric unit of the crystal, mediated largely through contacts in an N-terminal helix. Analytical ultracentrifugation and small-angle X-ray scattering confirm that StPGM forms a dimer in solution. Multiple sequence alignments and phylogenetic studies show that a distinct subset of bacterial PGMs share the signature dimerization helix, while other bacterial and eukaryotic PGMs are likely monomers. These structural, biochemical, and bioinformatic studies of StPGM provide insights into the large α-D-phosphohexomutase enzyme superfamily to which it belongs, and are also relevant to the design of inhibitors specific to the bacterial PGMs.
Proteins Structure Function and Bioinformatics 04/2011; 79(4):1215-29. DOI:10.1002/prot.22957 · 2.63 Impact Factor