Fragment based screens of the -D-phosphohexomutases as an initial step for inhibitor design

Abstract only availableEnzymes in the -D-phosphohexomutases superfamily frequently play a role in the biosynthesis of carbohydrates and glycolipids, critical for bacterial virulence and growth. -D-phosphohexomutases are being increasingly shown to play key roles in microbial infections, including enzymes from several major human pathogens. Successful inhibition of enzymes in the -D-phosphohexomutases superfamily may result in decreased bacterial growth rates, decreased virulence and greater susceptibility to traditional antibiotic treatment. As an initial step toward inhibitor design, we are conducting fragment-based screens of two structurally characterized members of the enzyme superfamily: P. aeruginosa phosphomannomutase/ phosphoglucomutase (PMM/PGM) and S. typhimurium phosphoglucomutase (PGM). Fragment based screening is a relatively new method that has been effectively used in the design of drug-like inhibitors for many systems. Crystals of PMM/PGM and PGM have been soaked with cocktails of small molecules (typical molecular weight 100-300 Da) to determine their sites of interaction with the proteins. X-ray diffraction data will be collected on these soaked crystals and the binding sites analyzed to determine the optimal interactions for effective inhibition.Life Sciences Undergraduate Research Opportunity Progra

Structural basis for substrate specificity of the alpha-D-phosphohexomutase superfamily

Abstract only availablePhosphoacetylglucosamine mutase (PAGM) is a human enzyme that is the key to the formation of the essential metabolite UDP-N-acetylglucosamine. Bacterial phosphoglucomutase (PGM) from Acetobacter xylinum catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PAGM and PGM are members of the alpha-D-phosphohexomutase superfamily which all catalyze intramolecular phosphoryl transfer on sugar substrates. These two analogs are similar in their mechanism, but dissimilar in their substrate specificity, not only to each other, but also to other well characterized (structurally and mechanistically) members of their superfamily. Protein expression and purification techniques were used to attempt to produce crystals to determine the three dimensional structures of human PAGM and bacterial PGM by X-ray diffraction in order to clarify the structural explanation for substrate specificity within the alpha-D-phosphohexomutase superfamily.Life Sciences Undergraduate Research Opportunity Progra

Expression and purification of AlgX in Pseudomonas aeruginosa

Abstract only availablePseudomonas aeruginosa is a Gram-negative bacterium that is the leading cause of hospital acquired infections. While this bacteria is present in water and soil, this bacteria only severely affects severely ill patients, such as those with cystic fibrosis. In cystic fibrosis patients, the bacteria will lodge in the lungs and form a coating, called a biofilm, around itself to protect it from the natural defenses of the body and antibiotics. This biofilm is formed by exopolysacchride sugar, known as alginate. The goal of this project is to, by isolating some of the proteins that help produce the biofilm, find the three-dimensional structure of those proteins. This will make it easier for various pharmaceutical research companies to create a drug to inhibit the specific active site in the proteins, preventing biofilm formation and allowing the infection to be treated with traditional antibiotics. The proteins encoded by two genes were picked for this study, algK and algX. Both are believed good targets because they have been shown in previously written papers to be critical to formation of the biofilm in P. aeruginosa. (1, 2) The cloned genes (in the form of a plasmid) previously obtained were used in the transformation step into E. coli. The lab protocol for general transformations was followed. To show that the bacteria were actually producing the protein we needed, the growth tubes were harvested; the cells lysed, and run on a 10% acrylamide gel to check for protein expression in the experimental versus control sample. The algK gel was inconclusive, however algX clearly overexpresses in large quantities. While most of the protein is insoluble, enough is soluble to warrant complete purification of the protein to set up crystallization trays. The current problem is how to optimize purification, because it co-elutes from a nickel column with contaminating proteins. A slow concentration bump of the buffers used to purify is being tested to see if it helps with this problem. References 1. Antonette Robles-Price, Thiang Yian Wong, Havard Sletta, Svein Valla, Neal L. Schiller. 2004. AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in Pseudomonas aeruginosa. J. of Bacteriology. 186:7369-7377. 2. Jain, Sumita, Franklin, Michael J., Ertesvag, Helga, Valla, Svein, Ohman, Dennis E. 2003. The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa. Molecular Microbiology. 47. 1123-1133NSF-REU Program in Biological Sciences & Biochemistr

A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

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

Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily

Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS) data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes

Role of the domain 4 rotation in PMM/PGM from P. aeruginosa during catalysis [abstract]

Abstract only availableThe enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) belongs to the alpha-D-phosphohexomutase enzyme superfamily. It catalyzes the reversible conversion of 1-phospho to 6-phospho sugars. The reaction mechanisms involves two phosphoryl transfers, with a 180° reorientation of the reaction intermediate during catalysis. Research performed by Regni et al. (2002) on the crystal structure of the protein shows that the enzyme has four domains arranged in a "heart" shape. Domains 1-3 share a similar tertiary fold and many interfaces with each other, while domain 4 is structurally unrelated and shares fewer interfaces to the other three domains. A comparison of the apo-protein and enzyme-substrate structures showed that there was a rotation of domain 4 upon ligand binding; the movement is located between residues 365 to 381 connecting domains 3 and 4. Based on the structural and evolutionary analysis we determined that the highly conserved residues(P368, S369, P369, Y17 and R262) all showed significant importance in the movement of the fourth domain upon ligand binding and were chosen for site-directed mutation. Alanine was chosen for all mutations in order to decrease the interactions between domain 4 and the rest of the enzyme. To see how the mutation effects the activity of the enzyme we will couple its reaction to that of glucose 6-phosphate dehydrogenase, pairing the formation of glucose 6-phosphate to NADH formation, and therefore monitoring the formation of NADH by its absorbance at 340 and comparing its activity to that of the wild-type. Catagorization of mutants P368 to Alanine and S369 to Alanine, have shown a decreased affinity of the enzyme to the substrate compared to that of the wild-type.Life Sciences Undergraduate Research Opportunity Progra

Role of the interdomain rotation of PMM/PGM from P. aeruginosa in catalysis [abstract]

Abstract only availableFaculty Mentor: Dr. Lesa Beamer, BiochemistryThe enzyme /phosphoglucomutase (PMM/PGM) belongs to the alpha-D-phosphohexomutase enzyme superfamily. It catalyzes the reversible conversion of 1-phospho to 6-phospho sugars. The reaction mechanism involves two phosphoryl transfers, with a 180° reorientation of the reaction intermediate during catalysis. The crystal structure of the protein shows that the enzyme has four domains arranged in a “heart” shape. Domains 1-3 share a similar tertiary fold and many interactions with each other, while domain 4 is structurally unrelated and has fewer interactions with the other three domains. A comparison of the apo-protein and enzyme-substrate structures showed that there was a rotation of domain 4 upon ligand binding; the movement is primarily localized between residues 365 to 381, which connect domains 3 and 4. Based on structural and evolutionary analysis we hypothesized that the highly conserved residues (P368, S369, P369, Y17 and R262) would be involved in the movement of the fourth domain upon ligand binding and these residues were selected for site-directed mutagenesis. These residues were mutated to alanine in order to change the interactions between domain 4 and the rest of the enzyme. To see how the mutation effects the activity of the enzyme, it will be assayed using a coupled reaction with glucose 6-phosphate dehydrogenase, that pairs the formation of glucose 6-phosphate to NADH formation, and can be monitored by measuring the formation of NADH by its absorbance at 340. By comparing the activity of mutants to wild-type enzyme, we see that the P368A and S369A mutants have a decreased affinity of the enzyme for substrate

Interallelic Complementation at the Ubiquitous Urease Coding Locus of Soybean

Soybean (Glycine max [L.] Merrill) mutant aj6 carries a single recessive lesion, aj6, that eliminates ubiquitous urease activity in leaves and callus while retaining normal embryo-specific urease activity. Consistently, aj6/aj6 plants accumulated urea in leaves. In crosses of aj6/aj6 by urease mutants at the Eu1, Eu2, and Eu3 loci, F(1) individuals exhibited wild-type leaf urease activity, and the F(2) segregated urease-negative individuals, demonstrating that aj6 is not an allele at these loci. F(2) of aj6/aj6 crossed with a null mutant lacking the Eu1-encoded embryo-specific urease showed that ubiquitous urease was also inactive in seeds of aj6/aj6. The cross of aj6/aj6 to eu4/eu4, a mutant previously assigned to the ubiquitous urease structural gene (R.S. Torisky, J.D. Griffin, R.L. Yenofsky, J.C. Polacco [1994] Mol Gen Genet 242: 404–414), yielded an F(1) having 22% ± 11% of wild-type leaf urease activity. Coding sequences for ubiquitous urease were cloned by reverse transcriptase-polymerase chain reaction from wild-type, aj6/aj6, and eu4/eu4 leaf RNA. The ubiquitous urease had an 837-amino acid open reading frame (ORF), 87% identical to the embryo-specific urease. The aj6/aj6 ORF showed an R201C change that cosegregated with the lack of leaf urease activity in a cross against a urease-positive line, whereas the eu4/eu4 ORF showed a G468E change. Heteroallelic interaction in F(2) progeny of aj6/aj6 × eu4/eu4 resulted in partially restored leaf urease activity. These results confirm that aj6/aj6 and eu4/eu4 are mutants affected in the ubiquitous urease structural gene. They also indicate that radical amino acid changes in distinct domains can be partially compensated in the urease heterotrimer