Cyclopropane-1,1-dicarboxylate is a slow-, tight-binding inhibitor of rice ketol-acid reductoisomerase

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of the branched-chain amino acids. The reaction catalyzed consists of two stages, the first of which is an alkyl migration from one carbon atom to its neighbour. The likely transition state is therefore a cyclopropane derivative, and cyclopropane-1,1-dicarboxylate(CPD) has been reported to inhibit the Escherichia coli enzyme. In addition, this compound causes the accumulation of the substrate of ketol-acid reductoisomerase in plants. Here, we investigate the inhibition of the purified rice enzyme. The cDNA was cloned, and the recombinant protein was expressed in E. coli, purified and characterized kinetically. The purified enzyme is strongly inhibited by cyclopropane-1,1-dicarboxylate, with an inhibition constant of 90 nM. The inhibition is time-dependent and this is due to the low rate constants for formation (2.63 X 10(5) M-1 min(-1)) and dissociation (2.37 x 10(-2) min(-1)) of the enzyme-inhibitor complex. Other cyclopropane derivatives are much weaker inhibitors while dimethylmalonate is moderately effective. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase

The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed. with its product, S-adenoSyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Va153, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased K-m values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its k(cat) value. Both WT hPNMT and D267A had similar k(cat) values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The K-i values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenoSyl-(L)-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.

Enzyme Electrochemistry - Biocatalysis on an electrode

Oxidoreductase enzymes catalyze single- or multi-electron reduction/oxidation reactions of small molecule inorganic or organic substrates, and they are integral to a wide variety of biological processes including respiration, energy production, biosynthesis, metabolism, and detoxification. All redox enzymes require a natural redox partner such as an electron-transfer protein ( e. g. cytochrome, ferredoxin, flavoprotein) or a small molecule cosubstrate ( e. g. NAD(P)H, dioxygen) to sustain catalysis, in effect to balance the substrate/product redox half-reaction. In principle, the natural electron-transfer partner may be replaced by an electrochemical working electrode. One of the great strengths of this approach is that the rate of catalysis ( equivalent to the observed electrochemical current) may be probed as a function of applied potential through linear sweep and cyclic voltammetry, and insight to the overall catalytic mechanism may be gained by a systematic electrochemical study coupled with theoretical analysis. In this review, the various approaches to enzyme electrochemistry will be discussed, including direct and indirect ( mediated) experiments, and a brief coverage of the theory relevant to these techniques will be presented. The importance of immobilizing enzymes on the electrode surface will be presented and the variety of ways that this may be done will be reviewed. The importance of chemical modification of the electrode surface in ensuring an environment conducive to a stable and active enzyme capable of functioning natively will be illustrated. Fundamental research into electrochemically driven enzyme catalysis has led to some remarkable practical applications. The glucose oxidase enzyme electrode is a spectacularly successful application of enzyme electrochemistry. Biosensors based on this technology are used worldwide by sufferers of diabetes to provide rapid and accurate analysis of blood glucose concentrations. Other applications of enzyme electrochemistry are in the sensing of macromolecular complexation events such as antigen - antibody binding and DNA hybridization. The review will include a selection of enzymes that have been successfully investigated by electrochemistry and, where appropriate, discuss their development towards practical biotechnological applications.

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH-could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH-with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the R2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT K-i = 1.3 mu M) is the most potent compound in this series and is quite selective for PNMT versus the R2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT K-i = 0.13 mu M). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH-is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Low mannose-binding lectin function is associated with sepsis in adult patients

Mannose-binding lectin (MBL) is an innate immune system pattern recognition molecule that kills a wide range of pathogens via the lectin complement pathway. MBL deficiency is associated with severe infection but the best measure of this deficiency is undecided. We investigated the influence of MBL functional deficiency on the development of sepsis in 195 adult patients, 166 of whom had bloodstream infection and 35 had pneumonia. Results were compared with 236 blood donor controls. MBL function (C4b deposition) and levels were measured by enzyme-linked immunosorbent assay. Using receiver-operator characteristics of MBL function in healthy controls, we identified a level of < 0.2 U mu L-1 as a highly discriminative marker of low MBL2 genotypes. Median MBL function was lower in sepsis patients (0.18 U mu L-1) than in controls (0.48 U mu L-1, P < 0.001). MBL functional deficiency was more common in sepsis patients than controls (P < 0.001). MBL functional deficient patients had significantly higher sequential organ failure assessment (SOFA) scores and higher MBL function and levels were found in patients with SOFA scores predictive of good outcome. Deficiency of MBL function appears to be associated with bloodstream infection and the development of septic shock. High MBL levels may be protective against severe sepsis. © 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3,site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins, beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Effect of polymorph derived oxidants on IgG in relation to rheumatoid factor binding

Immunoglobulin G from rheumatoid patients is denatured around the hinge region. This has been proposed as an explanation for the presence of circulating autoantibodies to IgG in these patients. It has previously been suggested that oxygen radicals (OR) derived from activated polymorphs may play a role in denaturation in vivo. Using sera from rheumatoid patients and age-matched controls in a modified ELISA technique, we have investigated the potential for polyclonal rheumatoid factors (RF) to bind to OR denatured IgG. Three model systems were used to generate OR in vitro: (a) purified PMN s activated by the cell surface stimulant PMA, (b) radiolysis of IgG in solution to generate specifically the superoxide radical and, in a separate system, the hydroxyl radical, (OH.), (c) purified myeloperoxide in the presence of H2O2 and halide ions. Results: 1. The binding of both IgA and IgM RF s to PMN denatured IgG increased dose dependently for seropositive sera only. 2. The OH. radical but not the superoxide radical significantly increased the binding of IgA and M RF, again only for seropositive sera. 3. The myeloperoxidase enzyme system did not increase RF binding. 4. IgG incubated with elastase was not found to be a better antigen than native IgG. These results indicate that IgG is denatured by OR released from activated PMN, thereby producing an antigen for polyclonal RF s.

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of small molecules to DNA junctions

Previous results in our laboratory suggest that the (CG) 4 segments whether present in a right-handed or a left-handed conformation form distinctive junctions with adjacent random sequences. These junctions and their associated sequences have unique structural and thermodynamic properties that may be recognized by DNA-binding molecules. This study probes these sequences by using the following small ligands: actinomycin D, 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione, ametantrone, and tris(phenanthroline)ruthenium (II). These ligands may recognize the distinctive features associated to the (CG)4 segment and its junctions and thus interact preferentially near these sequences. Restriction enzyme inhibition assays were used to determine whether or not binding interactions took place, and to approximate locations of these interactions. These binding studies are first carried out using two small synthetic oligomers BZ-III and BZ-IV. The (5meCG)4 segment present in BZ-III adopts the Z-conformation in the presence of 50 m M Co(NH3)63+. In BZ-IV, the unmethylated (CG)4 segment changes to a non-B conformation in the presence of 50 m M Co(NH3)63+. BZ-IV, containing the (CG)4 segment, was inserted into a clone plasmid then digested with the restriction enzyme Hinf I to produce a larger fragment that contains the (CG)4 segment. The results obtained on the small oligomers and on the larger fragment for restriction enzyme Mbo I indicate that 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione binds more efficiently at or near the (CG)4 segment. Restriction enzymes EcoRV, Sac I and Not I with cleavage sites upstream and downstream of the (CG)4 insert were used to further localize binding interactions in the vicinity of the (CG)4 insert. RNA polymerase activity was studied in a plasmid which contained the (CG)4 insert downstream from the promoter sites of SP6 and T7 RNA polymerases. Activities of these two polymerases were studied in the presence of each one of the ligands used throughout the study. Only actinomycin D and spider, which bind at or near the (CG)4 segment, alter the activities of SP6 and T7 RNA polymerases. Surprisingly, enhancement of polymerase activity was observed in the presence of very low concentrations of actinomycin D. These results suggest that the conformational features of (CG) segments may serve in regulatory functions of DNA. ^

N-acetoxy-N-acetyl-2-aminofluorene binding sites on [phi] X174 and SV40

Restriction enzyme inhibition and lambda exonuclease studies indicate that carcinogen N-acetoxy-N-acetyl-2 aminofluorene (AAAF) binds to sequences on ɸX174 RF and SV40 plasmids DNA that are similar to the eight preferred binding sites previously located on pBR 322. Both DNAs were digested with enzyme Hinf I and resultant fragments 32P end-labeled. Labeled fragments were reacted with the carcinogen to give one to sixteen bound moieties per DNA. Fragments were isolated and restriccion enzyme and lambda exonuclease inhibition assays were performed. Inhibition detected occurred at selected sites and was not specific for a certain enzyme or certain size of recognition sequence. Results of these assays allow mapping of the location of high affinity binding sites of the carcinogen on both DNAs. All sites have common sequence elements: the presence of either the sequence T(G/C)TT(G/C) or the sequence T(G/C) CTT(G/C).