Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses.

Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-ß-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-ß-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.

Geometric dependence of the conductance drop in a nanopore due to a particle.

We study the effect of a neutral particle on the ionic flow through a nanopore using a basic uniform field theory and the coupled Poisson-Nernst-Planck and Navier-Stokes (PNP-NS) equations. We consider hourglass and cylindrical pore profiles and examine how the difference in pore shape changes the position dependence of the current change due to the particle. Good quantitative agreement between both calculations is seen, though we find that the simple theory is unable to correctly capture the change in the access resistance of the pore if a particle is placed at the pore entrance. Finally, we examine the spatial variations in the solutions of the PNP-NS equations, finding that the electro-osmotic flow through the pore is completely disrupted for sufficiently large particles.

Cortically restricted production of IP3 leads to propagation of the fertilization Ca2+ wave along the cell surface in a model of the Xenopus egg.

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free cytosolic Ca2+ concentration that emanates from the point of sperm-egg fusion and traverses the entire diameter of the egg. This phenomenon appears to involve an increase in inositol-1,4,5-trisphosphate (IP3) resulting from interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. We have proposed models based on a static elevated distribution of IP3, and dynamic [IP3], however, these models have suggested that the fertilization wave passes through the center of the egg. Complementing these earlier models, we propose a more detailed model of the fertilization Ca2+ wave in Xenopus eggs to explore the hypothesis that IP3 is produced only at or near the plasma membrane. In this case, we find that the wave propagates primarily through the cortex of the egg, and that Ca2+ -induced production of IP3 at the plasma membrane allows IP3 to propagate in advance of the wave. Our model includes Ca2+ -dependent production of IP3 at the plasma membrane and IP3 degradation. Simulations in 1 dimension and axi-symmetric 3 dimensions illustrate the basic features of the wave.