Promotion of enzyme flexibility by dephosphorylation and coupling to the catalytic mechanism of a phosphohexomutase.
ABSTRACT: The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.
Project description:Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates. We compared states the enzyme visits during its catalytic cycle. Collective responses of Pseudomonas PMM/PGM to phosphosugar substrates and inhibitor were assessed using NMR-detected titrations. Affinities were estimated from binding isotherms obtained by principal component analysis (PCA). Relationships among phosphosugar-enzyme associations emerge from PCA comparisons of the titrations. COordiNated Chemical Shifts bEhavior (CONCISE) analysis provides novel discrimination of three ligand-bound states of PMM/PGM harboring a mutation that suppresses activity. Enzyme phosphorylation and phosphosugar binding appear to drive the open dephosphorylated enzyme to the free phosphorylated state, and on toward ligand-closed states. Domain 4 appears central to collective responses to substrate and inhibitor binding. Hydrogen exchange reveals that binding of a substrate analogue enhances folding stability of the domains to a uniform level, establishing a globally unified structure. CONCISE and PCA of NMR spectra have discovered novel states of a well-studied enzyme and appear ready to discriminate other enzyme and ligand binding states.
Project description:The alpha-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates. The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans. Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition. Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the alpha-D-phosphohexomutase superfamily. These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P. aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family. Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family. In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.
Project description: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.
Project description:A domain needed for the catalytic efficiency of an enzyme model of simple processivity and domain-domain interactions has been characterized by NMR. This domain 4 from phosphomannomutase/phosphoglucomutase (PMM/PGM) closes upon glucose phosphate and mannose phosphate ligands in the active site, and can modestly reconstitute activity of enzyme truncated to domains 1-3. This enzyme supports biosynthesis of the saccharide-derived virulence factors (rhamnolipids, lipopolysaccharides, and alginate) of the opportunistic bacterial pathogen Pseudomonas aeruginosa. (1)H, (13)C, and (15)N NMR chemical shift assignments of domain 4 of PMM/PGM suggest preservation and independence of its structure when separated from domains 1-3. The face of domain 4 that packs with domain 3 is perturbed in NMR spectra without disrupting this fold. The perturbed residues overlap both the most highly coevolved positions in the interface and residues lining a cavity at the domain interface.
Project description:Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an ?-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ?-strands within the (?/?)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.
Project description:Glucokinase phosphorylated a series of C-1 fluorinated ?-d-gluco-heptuloses. These phosphorylated products were discovered to be inhibitors of ?-phosphomannomutase/phosphoglucomutase (?PMM/PGM) and ?-phosphoglucomutase (?PGM). Inhibition potency with both mutases inversely correlated to the degree of fluorination. Structural analysis with ?PMM demonstrated the inhibitor binding to the active site, with the phosphate in the phosphate binding site and the anomeric hydroxyl directed to the catalytic site.
Project description:Two complexes of the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa with a slow substrate and with an inhibitor have been characterized by X-ray crystallography. Both ligands induce an interdomain rearrangement in the enzyme that creates a highly buried active site. Comparisons with enzyme-substrate complexes show that the inhibitor xylose 1-phosphate utilizes many of the previously observed enzyme-ligand interactions. In contrast, analysis of the ribose 1-phosphate complex reveals a combination of new and conserved enzyme-ligand interactions for binding. The ability of PMM/PGM to accommodate these two pentose phosphosugars in its active site may be relevant for future efforts towards inhibitor design.
Project description:?-Phosphoglucomutase (?PGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of ?-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a ?-glucose 1,6-bisphosphate intermediate. Substrate-free ?PGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into ?PGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free ?PGMWT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1H, 15N and 13C backbone resonance assignments of the P146A variant (?PGMP146A) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of ?PGMWT, where the K145-P146 peptide bond is cis. In ?PGMP146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect ?PGM crystal structures, and a chemical shift comparison between substrate-free ?PGMP146A and ?PGMWT confirms that the solution conformations are very similar, except for the D137-A147 loop. Hence, the isomerisation state of the 145-146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12-T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114-S116), and the D137-A147 loop (V141-A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic MgII ion and the L44-L53 loop responsible for substrate discrimination.
Project description:The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.
Project description:Four orthologous genes (TK1108, TK1404, TK1777, and TK2185) that can be annotated as phosphomannomutase (PMM) genes (COG1109) have been identified in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. We previously found that TK1777 actually encodes a phosphopentomutase. In order to determine which of the remaining three orthologues encodes a phosphoglucomutase (PGM), we examined the PGM activity in T. kodakaraensis cells and identified the gene responsible for this activity. Heterologous gene expression and purification and characterization of the recombinant protein indicated that TK1108 encoded a protein with high levels of PGM activity (690 U mg(-1)), along with high levels of PMM activity (401 U mg(-1)). Similar analyses of the remaining two orthologues revealed that their protein products exhibited neither PGM nor PMM activity. PGM activity and transcription of TK1108 in T. kodakaraensis were found to be higher in cells grown on starch than in cells grown on pyruvate. Our results clearly indicate that, among the four PMM gene orthologues in T. kodakaraensis, only one gene, TK1108, actually encodes a protein with PGM and PMM activities.