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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.09.010 Byline: Jacopo F. Novelli (a), Kshitiz Chaudhary (a), Julie Canovas (a), Jack S. Benner (a), Catherine L. Madinger (a), Paul Kelly (c), Jonathan Hodgkin (b), Clotilde K.S. Carlow (a) Keywords: C. elegans; Galactofuranose; UGM; Surface coat; Glycans; M. nematophilum; Nematodes Abstract: Galactofuranose (Gal.sub.f), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal.sub.f biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal.sub.f in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal.sub.f in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization-mass spectrometry (ESI-MS). UDP-Gal.sub.f was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal.sub.f likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species. Author Affiliation: (a) Division of Molecular Parasitology, New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA (b) Department of Biochemistry, Genetics Unit, South Parks Road, OX1 3QU Oxford, UK (c) Salem State College, 352 Lafayette Street, Salem, MA 01970, USA Article History: Received 30 March 2009; Revised 15 August 2009; Accepted 7 September 2009

The synthesis and evalutation of cyclopropyl and epoxy substrate analogues which are mechanism-based irreversible inhibitors is reported. The evaluation is carried out by type 2 isopentenyl diphosphate isomerase (IDI-2) from Thermus thermophilus.

The enzyme 2- hydroxychromene-2-carboxylix acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion ([K.sub.eq] =1.5) of HCCA and trans-a-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The results suggests that isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate.

The crystal structure of yeast protein disulfide isomerase reveals that the four thioredoxin domains are arranged in the shape of a twisted 'U' with the active sites facing each other across the long sides of the 'U'. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

A modular total synthesis of lamellarin D, a marine alkaloid with potent cytotoxic as well as topoisomerase I inhibition properties, are accomplished. A sequential and regioselective bromination/Suzuki cross-coupling procedure was applied for the introduction of aryl groups.

Several computational studies are conducted to analyze the different properties exhibited during the hydrogen bonding taking place in the active site of ketosterois isomerase. The changes taking place in the NMR chemical shifts and electronic absorption spectra of the system due to the bonding are also discussed.

Parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPlase), has physically interacted with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, which is determined by affinity chromatography and chemical cross-linking experiments. The studies have shown that Par10 catalytic activity is involved in the cellular protection against oxidative stress.

Redox-dependent conformational and solvation changes of protein disulfide isomerase (PDI) from a thermophilic fungus are studied by using small-angle X-ray scattering (SAXS) analysis. The results have shown that the conformation of PDI is controlled by the redox state of the active site cysteine residues in the thioredoxin a and a' domains and that the conformational alternation has accompanied solvation changes in the active site cleft formed by the thioredoxin a, b, b' and a' domains.

Human nuclear Cyclophilin 33 (CYP33) has mediated downregulation of the expression of mixed lineage leukemia (MLL) target genes HOXC8, HOXA9, CDKN1B and C-MYC, in a proline isomerase-dependent manner. The results have provided a mechanism for the MLL plant homeodomain 3 (PHD3) domain in order to act as a switch between activation and repression.

A novel chemical tool to exclusively populate native disulfide-containing intermediates in unstructured forms was applied to determine the impact of protein disulfide isomerase (PDI) and its subdomains on the rate-determining step in ribonuclease A folding and on the physical structure-forming step of select endoplasmic reticulum (ER)-processed proteins including RNase A. Analysis of folding kinetics of RNase A and other proteins provide insight into the highly evolved oxidoreductase activity of PDI which masks its chaperone-like activity, impedes conformational folding of ER-processed proteins, and limits its potential to accelerate the rate-determining step in oxidative regeneration.

The syntheses of allene and alkyne substrate analogues for the enzyme isopentenyl diphosphate isomerase (IDI) are carried out to study the mechanism of isomerization for type II enzyme (IDI-2). The experimental and computational results are consistent with a protonation-deprotonation mechanism for the IDI enzyme-catalyzed isomerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules.

The various characteristics associated with the proton affinity of the oxyanion hole present in the active site of ketosteroid isomerase are discussed. The strength of the hydrogen bond in the compound is shown to increase with a decrease in the photon affinity.

A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual residues was used to study the molecular determinants for the catalytic efficiency of Bacillus licheniformis L-arabinose isomerase (L-AI). The data obtained and molecular dynamics simulations indicated that one of the conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. lichenifromis L-AI is involved in the catalytic efficiency of B. licheniformis L-AL.

A study related to the single-domain cyclophilin 18 (Cyp18), known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and also function as both a folding catalyst and a chaperone is presented. The chaperone effect of Cyp18 demonstrated for citrate synthase indicated a general chaperone effect of Cyp18 PPI.

A gene encoding a glycoside hydrolase family 44 (GH44) proteins from Clostridium acetobutylicum ATCC 824 is prepared and transformed into Escherichia coli. Crystallization and X-ray diffraction have shown a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and [beta]-sandwich folds, which is similar to other GH44 crystal structures.

A library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus are synthesized to fully explore the substrate specificities of prolyl isomerases. The peptide library could be used to provide a complete set of P1-site specificities for prototypic human members of the three prolyl isomerase families, FKBP12, cyclophilin 18, and parvulin 14.

The characterization and the properties of the various enzymes, L-arabinose and mannose-6-phosphate isomerase obtained from Geobacilus thermodenitrificans are discussed. These enzymes are shown to be extremely useful in the production of L-ribose.

The gene encoding mannose-6-phosphate isomerase from Bacillus subtilis is cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme for various aldoses and ketoses is examined. L-Ribulose has displayed the highest activity among all pentoses and hexoses and hence mannose-6-phosphate isomerase is applied to the production of L-ribose from L-ribulose.

NMR spectroscopy is used to determine the solution structure of oxidized ERp18, the smallest member of the protein disulfide isomerase (PDI) family of proteins to contain a Cys-Xxx-Xxx-Cys active site motif. The results confirmed a putative role for ERp18 within the cell as an oxidase, introducing disulfide bonds to substrate proteins, and also provide structural confirmation for role of ERp18 as a thiol-disulfide oxidoreductase.

Nucleic acid databases for sequences encoding putative xylose isomerase (XIs) were screened to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in Saccharomyces cerevisiae. The ability of the bacterial XI expression to grow on xylose and ferment xylose to enthanol could find potential application as industrially useful yeast strain to improve xylose fermentation.

Several studies are conducted to explain the structural and functional characterization of the phosphoglycerate mutase (PGM) domain of Sts, a type of protein tyrosine phosphatase (PTP). The different Sts structures are shown to exhibit an acid-dependent phosphatase activity, which is highly affected by the active site residues.

The crystal structure of the ecdysone phosphate phosphatase (EPPase) phosphoglycerate mutase (PGM) homology domain is described. The results have shown a new protein tyrosine phosphatase (PTP) activity for EPPase, suggesting that EPPase and its closest homologues can be grouped into a distinct subfamily in the large 2H-phosphatase superfamily of proteins.

Different methods are used to characterize the thermal dissociation and unfolding of the triosephosphate isomerase (TIM) from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). The results showed that the temperature-induced unfolding of EhTIM is a multistate process with at least two transitions, one which involves the reversible dissociation of the native state into a monomeric intermediate and other related to monomer unfolding and linked to irreversible protein aggregation.

A mutational approach of histidine H59 and H157 was used on peptidyl-prolyl cis/trans isomerase (Pin1) function, activity, and protein stability in order to define the sequence requirements at positions H59 and H157 for hydrogen bonding network in the active site. The results revealed that the H59 and H157 residues are not important for hydrogen bonding or direct interactions with the substrate, but play a key role in the structure and stability of the active site.

The studies of stabilized N-terminal region in the crystal structure of Thermus thermophilus type 2 isopentenyl diphosphate isomerase in complex with inorganic pyrophosphate are reported. The results provide insight into the putative active site and the catalytic mechanism, which stabilize the enzyme, isopentenyl diphosphate isomerase 2 similar to protonation-deprotonation mechanism of isopentenyl diphosphate isomerase 1.

The two Arg residues, [Arg.sup.325] and [Arg.sup.328] for isomaltose production was explored to gain insight into the significant role of the mutants in sucrose isomerase (SI)-substrate binding in Protaminobacter rubrum. The predicted fructose-binding site (FBS) three dimensional structure of SI shed light on the relationship between the enzyme activity and the transition state energy, which have great potential for enhancing isomaltose production.

A study was conducted to explore the crucial role of the testes-specific peptidyl-propyl cis/trans isomerase FKBP36 in male meiosis and the catalytic domains of FKBP36, which bind to clathrin heavy chain (CHC) of clathrin. The results from identified interactions among clathrin-FKBP36-Hsp72 complexes which bind to the matrices of clathrin-coated vesicles in spermatocytes revealed the possible role of FKBP36 and Hsp72 in the disassembly of clathrin coats.

DNA cleavage assays, saturation transfer difference [super 1]H NMR spectroscopy, and enzyme-drug binding studies were conducted to define interactions between etoposide and human topoisomerase II[alpha]. The results suggest that the binding of etoposide to human topoisomerase II[alpha] is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring.

Ultraviolet visible (UV-vis) spectral analysis of the type II isopentenyl diphosphate/dimethyallyl diphosphate isomerase (IDI-2) reaction has shown the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme is incubated with isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP). The results have shown that the C2-H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis is involved during turnover.

The unusual mechanism for a phenylalanine aminomuatse (PAM) rearrangement was studied by isotopic labeling. The results established that the amino group at C2 and the vicinal pro-3S hydrogen are interchanged by PAM with retention of configuration at both migration termini.

The binding of phenolate transition state analogues (TSAs) to ketosteroid isomerase (KSI) shows that the electrostatic contributions to binding of TSAs can be very different from the corresponding contributions to the catalysis. The studies reveal that the electrostatic preorganization is highly dependant on the orientation of the nonpolar form of the TSAs relative to the oxyanion hole.

E376 mutants are constructed, and mutant enzymes are purified and characterized. The results indicate that E376 is the catalytic residue for both dehydrgenase and isomerase activities of the enzyme.

The effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. The finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.