Biomedical subjects

Geoffrey B Fincher

Publications and source records attributed to Geoffrey B Fincher.

28 records · Page 2Linked to original sources

PubMed2003

Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic.

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and 4H3 conformer mimic of a glucoside, binds very tightly to a barley beta-d-glucan glucohydrolase, with a Ki constant of 2 x 10(-9) m and a DeltaG of 51 kJ mol(-1). PheGlcIm binds to the barley beta-d-glucan glucohydrolase approximately 2 x 10(5) times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10(6) times tighter than 4-nitrophenyl beta-d-glucopyranoside, and 2 x 10(7) tighter than glucose. The three-dimensional structure of the beta-d-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 sigma level form between the three active site residues Asp95, His207, and Asp285, and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the beta-d-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 A. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the 4E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.

Catalytic Domain↗

PubMed2003

An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation.

Arabidopsis was transformed with double-stranded RNA interference (dsRNAi) constructs designed to silence three putative callose synthase genes: GLUCAN SYNTHASE-LIKE5 (GSL5), GSL6, and GSL11. Both wound callose and papillary callose were absent in lines transformed with GSL5 dsRNAi and in a corresponding sequence-indexed GSL5 T-DNA insertion line but were unaffected in GSL6 and GSL11 dsRNAi lines. These data provide strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation. Deposition of callosic plugs, or papillae, at sites of fungal penetration is a widely recognized early response of host plants to microbial attack and has been implicated in impeding entry of the fungus. Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis. Paradoxically, the absence of callose in papillae or haustorial complexes correlated with the effective growth cessation of several normally virulent powdery mildew species and of Peronospora parasitica.

Arabidopsis↗

PubMed2003

Synthesis of complex oligosaccharides by using a mutated (1,3)-beta-D-glucan endohydrolase from barley.

Complex oligosaccharides with newly formed (1,3)-beta-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted alpha-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide beta-D-hexopyranoside acceptors by using a (1,3)-beta-D-glycosynthase. These linear and branched (1,3)-beta-linked oligosaccharides could prove to be important in a range of medical, pharmaceutical, and agricultural applications. Furthermore, the observation that the (1,3)-beta-D-glucan glycosynthase accommodates (1,3)-, (1,4),- and (1,6)-beta-oligosaccharides in its acceptor subsites suggests novel, yet unexpected physiological roles for the wild type (1,3)-beta-D-glucan endohydrolase from higher plants.

Amino Acid Substitution↗

PubMed2003

Biochemical evidence linking a putative callose synthase gene with (1 --> 3)-beta-D-glucan biosynthesis in barley.

A putative barley (1 --> 3)-beta-D-glucan synthase cDNA of 6.1 kb, which is homologous to the yeast FKS gene, was assembled from DNA fragments obtained through screening of barley cDNA and BAC libraries, and by PCR amplification. The corresponding gene, designated HvGSL1, is a member of a family of at least six genes in barley. Gene transcripts are detected at relatively high levels in early developing grain, florets, coleoptiles and roots, but not in leaves infected with a fungal pathogen. A (1 --> 3)-beta-D-glucan synthase has been purified more than 60-fold from barley suspension-cultured cells by detergent extraction, CaCl2 treatment, sucrose density gradient centrifugation and non-denaturing gel electrophoresis. The enzyme synthesizes (1 --> 3)-beta-D-glucan in vitro and is recognized by antibodies raised against a 17 kDa protein generated by heterologous expression of a fragment of the HvGSL1 cDNA. Furthermore, mass spectrometric analyses show that tryptic peptides produced by in-gel digestion of the active enzyme match peptides predicted from the gene sequence. Thus, the amino acid sequence predicted from the HvGSL1 gene has been linked with the actual amino acid sequence of an active (1 --> 3)-beta-D-glucan synthase fraction from barley.

Amino Acid Sequence↗

PubMed2002

Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.

An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4'-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4'-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4'-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --> 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --> 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --> 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.

Amino Acid Sequence↗

PubMed2002

Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans.

Barley (1,3)-beta-D-glucan endohydrolases (EC ), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with alpha-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K(m)(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) m(-1) s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.

Base Sequence↗

PubMed2002

Induction of (1-->3,1-->4)-beta-D-glucan hydrolases in leaves of dark-incubated barley seedlings.

When seedlings of barley (Hordeum vulgare L.) were transferred from a natural light/dark cycle into darkness, (1-->3,1-->4)-beta- D-glucan endohydrolase (EC 3.2.1.73) activity in leaf extracts increased 3- to 4-fold after 2 days. Activity decreased to normal levels within a day if the light/dark cycle was restored. Although there are two (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzymes in barley, the increased enzyme activity in dark-grown seedlings was attributable entirely to increases in isoenzyme EI. Northern hybridization analyses confirmed that mRNA transcripts encoding (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzyme EI accumulated in the leaves of dark-incubated seedlings; no isoenzyme EII mRNA was detected. Activity of beta-D-glucan glucohydrolases also increased 10-fold after 2 days of dark treatment. The latter, broad-specificity enzymes release glucose from (1-->3,1-->4)-beta-D-glucans and from beta-D-oligoglucosides released by (1-->3,1-->4)-beta-D-glucan endohydrolases. Consistent with the activity patterns of these enzymes, the (1-->3,1-->4)-beta-D-glucan content of leaf cell walls decreased by about 30% when barley seedlings were transferred into darkness. Soluble sugars in the leaves decreased by about 60% during the same period. Because no measurable leaf elongation was detected during the various light/dark treatments, the enzymes were unlikely to be participating in wall loosening and cell elongation. Instead, the results suggest that cell wall (1-->3,1-->4)-beta-D-glucans can be re-mobilized in the non-elongating, dark-incubated leaves and the glucose so generated could serve as an energy source under conditions of sugar depletion.

Base Sequence↗

PubMed2002

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity.

Two mutant lines of barley, Risø 17 and Notch-2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen-accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A-type or B-type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild-type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild-type endosperm plays a role in determining the number, and hence the form, of starch granules.

Alleles↗

PubMed2002

Characterization of the genes encoding the cytosolic and plastidial forms of ADP-glucose pyrophosphorylase in wheat endosperm.

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified.

Amino Acid Sequence↗

PubMed2002

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases.

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.

Amino Acid Sequence↗