Inhibition of pectin methyl esterase activity by green tea catechins.

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

Effects of a distal mutation on active site chemistry.

Previous studies of Escherichia coli dihydrofolate reductase (ecDHFR) have demonstrated that residue G121, which is 19 A from the catalytic center, is involved in catalysis, and long distance dynamical motions were implied. Specifically, the ecDHFR mutant G121V has been extensively studied by various experimental and theoretical tools, and the mutation's effect on kinetic, structural, and dynamical features of the enzyme has been explored. This work examined the effect of this mutation on the physical nature of the catalyzed hydride transfer step by means of intrinsic kinetic isotope effects (KIEs), their temperature dependence, and activation parameters as described previously for wild type ecDHFR [Sikorski, R. S., et al. (2004) J. Am. Chem. Soc. 126, 4778-4779]. The temperature dependence of initial velocities was used to estimate activation parameters. Isotope effects on the preexponential Arrhenius factors, and the activation energy, could be rationalized by an environmentally coupled hydrogen tunneling model, similar to the one used for the wild-type enzyme. Yet, in contrast to that in the wild type, fluctuations of the donor-acceptor distance were now required. Secondary (2 degrees ) KIEs were also measured for both H- and D-transfer, and as in the case of the wild-type enzyme, no coupled motion was detected. Despite these similarities, the reduced rates, the slightly inflated primary (1 degrees ) KIEs, and their temperature dependence, together with relatively deflated 2 degrees KIEs, indicate that the potential surface prearrangement was not as ideal as for the wild-type enzyme. These findings support theoretical studies suggesting that the G121V mutation led to a different conformational ensemble of reactive states and less effective rearrangement of the potential surface but has an only weak effect on H-tunneling.

Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly.

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.

Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate.

The interaction of dihydrofolate (H(2)F) and NADPH with a fluorescent derivative of H(2)F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H(2)F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 x 10(7) M(-1).s(-1), and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H(2)F with a bimolecular rate constant of 2.6-5.7 x 10(6) M(-1).s(-1), with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s(-1) at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme-substrate complexes exist.

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers.

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.

Bis(dimesitylmethylene)-1,3-dithietane, -1,2,4-trithiane, and -1,2,4,5-tetrathiane. Conformation and DNMR Study. Observable Dimesityl Thioketene.

The reaction of dimesityl ketene with P(2)S(5) and pyridine gives 2,4-bis(dimesitylmethylene)-1,3-dithietane (3), 3,6-bis(dimesitylmethylene)-1,2,4,5-tetrathiane (4), 3,5-bis(dimesitylmethylene)-1,2,4-trithiane (5), and dimesityl thioketene 2 as a transient. The structures of 3 and 4 were determined by X-ray crystallography. The dimesitylmethylene moieties in 3 and 4 have a propeller conformation, and the tetrathiane ring in 4 has a twist-boat conformation. Static NMR data are consistent with the presence of two enantiomers and one meso form for 3 and of three pairs of enantiomers for 4. The several aromatic signals observed for 3 and 4 at slow exchange at 160 K coalesce to a single signal at higher temperatures. The threshold barriers for these dynamic processes are 12.7 (3) and 13.3 (4) kcal mol(-)(1), and the dynamic behavior was analyzed in terms of flip processes. On standing, a solution of 3 develops a blue color which is attributed to formation of 2, by retro-dimerization of 3. Diphenylacetyl chloride gives with P(2)S(5) the analog of 5 and its one-double bond reduction product. Ditipylketene forms a product identified tentatively as the analog of 3.