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.

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.

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.

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.

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.

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.

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.