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One of the best systems for exploring the origin of enzyme catalysis has been the reaction of ketosteroid isomerase (KSI). Studies of the binding of phenolates to KSI have been taken as proof that the electrostatic preorganization effect only makes a minor contribution to the binding of the real, multiring, transition state (TS). However, our simulation study has determined that the difference between the phenolates and the TS arises from the fact that the nonpolar state of the phenolate can rotate freely relative to the oxyanion hole and thus loses the preorganization contribution. A recent study explored the reactivity of both small and multiring systems and concluded that their similar reactivity contradicts our preorganization idea. Herein, we establish that the available experiments in fact provide what is perhaps the best proof and clarification of the preorganization idea and its crucial role in enzyme catalysis. First, we analyze the binding energy and the [pK.sub.a] of equilenin and identify direct experimental evidence for our prediction about the differential electrostatic stabilization of the large TS and the small phenolates. Subsequently, we show that the similar reactivity of the small and large systems is also due to an electrostatic preorganization effect but that this effect only appears in the intermediate state because the TS is not free to rotate. This establishes the electrostatic origin of enzyme catalysis. We also clarify the crucial importance of having a well-defined physical concept when examining catalytic effects and the need for quantitative tools for analyzing such effects. electrostatic reorganization | empirical valence bond | enzyme catalysis | transition state analogue doi/10.1073/pnas.0914579107

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

Epoxy, diene and fluorinated substrate analogues, irreversible inhibitors of type I isopentenyl diphosphate isomerase (IDI-1), are explored as mechanistic probed for type II enzyme (IDD-2). The results have shown that the irreversible inhibitors inactivate the reduced flavin needed for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.

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 DNA damage checkpoint response delays cell cycle progression upon DNA damage and prevents genomic instability. Genetic analysis has identified sensor, mediator, signal transducer, and effector components of this global signal transduction pathway. Here we describe an in vitro system with purified human check-point proteins that recapitulates key elements of the DNA damage checkpoint. We show that the damage sensor ATR in the presence of topoisomerase II binding protein 1 (TopBP1) mediator/adaptor protein phosphorylates the Chk1 signal-transducing kinase in a reaction that is strongly dependent on the presence of DNA containing bulky base lesions. The dependence on damaged DNA requires DNA binding by TopBP1, and, indeed, TopBP1 shows preferential binding to damaged DNA. This in vitro system provides a useful platform for mechanistic studies of the human DNA damage checkpoint response. Chk1 kinase | damage recognition | signal transduction | topoisomerase II binding protein 1

It is often difficult to determine which of the sequence and structural differences between divergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from Escherichia coli. Surprisingly, we found single amino acid substitutions in DsbG that were able to complement dsbC in vivo and have more DsbC-like isomerase activity in vitro. Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over [approximately equal to] 0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure. chaperone | protein folding | directed evolution

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 phosphodiester backbone of DNA for the presence of strong topoisomerase II cleavage site is investigated using molecular dynamics simulations in explicit water and in the presence of sodium ions. The proximity of phosphate group to the cleavage site renders it the cleavage specificity to topoisomerase II.

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.

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), involving protonation of the carbon-carbon double bond to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP and IPP are synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme.

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.

A study is conducted on delta(super 5)-3-ketosteroid isomerase (KSI) catalysis, so molecular dynamics replicates the KSI dimer ligated with a substrate and the effect of the mediating analogue leading to increased ab initio computations on the significant enzymatic reaction of the samples. Finally, these computations show that the cooperative hydrogen bond method might be preffered to the catalytic diad method in the catalytic process of KSI.

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.

A method to identify and correct the departures from the Stark model due to direct hydrogen bonding based on comparison of [super 13]C NMR chemical shifts and IR frequencies each calibrated in turn by a solvatochromic model is presented. The experimental observations could be applied to a thiocyanate-modified protein, ketosteroid isomerase to demonstrate that the changes in electrostatic field could be measured within the complex protein environment even in the background of direct hydrogen bonding to the probe.

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.

Molecular dynamics simulations based on an empirical valence bond model are used to examine the impact of mutating residues Tyr14 and Asp99 in the active site of ketosteroid isomerase (KSI) on the hydrogen-bonding patterns, conformational changes, and van der Waals and electrostatic interactions during the proton transfer reactions. The results revealed that small conformational changes due to stochastic thermal motions are required within the preorganized active site formed by KSI to facilitate the proton transfer reactions.

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.

Molecular dynamics simulations are used for probing the properties of several anticancer alkaloid camptothecin and its analogs (CPTs) that form ternary complexes of greatly variable persistence. The correlated motions have primarily occurred between the CPTs and the flanking base pairs.

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 related to the packing and binding interactions within the keto-steroid isomerase (KSI) active site that could constrain local side-chain reorientation and prevent hydrogen bond shortening is reported. The findings from the studies provide evidence for subtle geometric effects that could have significant energetic consequences.

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.

The application of ketosteroid isomerase (KSI) in enhancing the motion/quantum mechanical hydrogen tunneling (HT) in enzymatic systems is discussed. KSI is shown to enhance the contribution of coupled motion (CM)/HT in the enzymatic reaction.

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.

The homo- and heterotrimetallic Zn(II) and Cu(II) complexes, [M(sub 2)M'{(D-Glc)(sub 2)-tacn}(sub 2)(XDK)]Cl(sub 2), prepared by stepwise construction from the D-glucose-binding mononuclear complexes, and [M{(D-Glc)(sub 2)-tacn}Cl]Cl, where tacn is 1,4,7-triazacyclononane is reported. These structures are useful substrate binding models of xylose or glucose isomerases, which promote D-glucose D-fructose isomerization by using divalent dimetallic centers bridged by a glutamate residue.

The enzyme isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase catalyzes the of isopentenyl pyrophospate to form dimethylallyl pyrophosphate, which is used in protein prenylation and in cholesterol and ergosterol biosynthesis. The structure of the enzyme is determined because the enzyme is a potential drug target.