Jelle Lahnstein

Bifunctional family 3 glycoside hydrolases from Barley with alpha-L-Arabinofuranosidase and beta-D-Xylosidase activity characterization, primary structures and COOH-terminal processing

An α-l-arabinofuranosidase and a β-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 α-l-arabinofuranoside is only slightly higher than during hydrolysis of 4′-nitrophenyl β-d-xyloside. Thus, the enzyme is actually a bifunctional α-l-arabinofuranosidase/β-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 β-d-xyloside, with a catalytic efficiency ∼30-fold higher than with 4′-nitrophenyl α-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 → 4)-β-d-xylan endohydrolase. Both enzymes hydrolyze (1 → 4)-β-d-xylopentaose, and ARA-I can also degrade (1 → 5)-α-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.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-β-d-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-β-d-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-β-d-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-β-dglucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-β-d-glucan:xyloglucan conjugate by a specific (1,3;1,4)-β-d-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus.

The C‐type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D‐amino acid at position 2 in the primary structure. Peptides containing a D‐amino acid have been found in lower forms of organism, but this report is the first for a D‐amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L‐amino acid residue in the protein to the D‐configuration.

Hydrolysis of (1,4)-β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-β-D-mannan endohydrolase and β-D-mannosidase

A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.

Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.)

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-beta-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The K(m) values were 40 microM for UDP-Gal and 55 muM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.

Separation and purification of soluble polymers and cell wall fractions from wheat, rye and hull less barley endosperm flours for structure-nutrition studies

The nutritional values associated with the cell walls of cereal endosperm flours are due to a combination of solubilized arabinoxylan and (1-3,1-4)-β-d-glucan as well as residual nonsolubilized cell wall material. In order to investigate structure-nutrition relationships, an appropriate method for the complete functional and structural characterization of cell wall polysaccharides in various cereal endosperm flours is described. This involves the separation of soluble polymers and the residual cell wall fraction without using organic solvents, and the fractionation of soluble polymers into arabinoxylan- and (1-3,1-4)-β-d-glucan-rich fractions for subsequent analysis. This methodology is applied to endosperm flours from wheat, hull-less barley and rye, and could be extended to include studies on the effects of food processing with respect to yield and characteristics of the three fractions in order to better understand the structural basis for nutritional functionality.

Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like—rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47–50% w/w) and non-cellulosic polysaccharides (16–22% w/w), and whole leaves were low in lignin (9–13% w/w). Of the dry mass of whole Agave leaves, 85–95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41–48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.

Characterization and Expression Patterns of UDP-d-Glucuronate Decarboxylase Genes in Barley

UDP-d-glucuronate decarboxylase (EC 4.1.1.35) catalyzes the synthesis of UDP-d-xylose from UDP-d-glucuronate in an essentially irreversible reaction that is believed to commit glycosyl residues to heteroxylan and xyloglucan biosynthesis. Four members of the barley (Hordeum vulgare) UDP-d-glucuronate decarboxylase gene family, designated HvUXS1 to HvUXS4, have been cloned and characterized. Barley HvUXS1 appears to be a cytosolic enzyme, while the others are predicted to be membrane-bound proteins with single transmembrane helices. Heterologous expression of a barley HvUXS1 cDNA in Escherichia coli yields a soluble enzyme that converts UDP-d-glucuronate to UDP-d-xylose, is associated with a single molecule of bound NAD+, and is subject to feedback inhibition by UDP-d-xylose. Quantitative PCR shows that the HvUXS1 mRNA is most abundant among the 4 HvUXS genes, accounting for more than 80% of total HvUXS transcripts in most of the tissues examined. The abundance of HvUXS1 mRNA is 10-fold higher in mature roots and stems than in leaves, developing grains, or floral tissues. Transcriptional activities of HvUXS2 and HvUXS4 genes are relatively high in mature roots, coleoptiles, and stems compared with root tips, leaves, and floral tissues, while HvUXS3 mRNA is low in all tissues. In barley leaf sections, levels of the most abundant mRNA, encoding HvUXS1, reflect the amount of soluble enzymic protein and activity. In selected tissues where HvUXS1 transcript levels are high, cell walls have higher arabinoxylan contents.

Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue

Abstract: The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the β, γ, δ, and ε chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.

Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)

A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400‐fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full‐length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post‐translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33–35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo‐xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme’s active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping‐pong (Bi, Bi) rather than a sequential reaction mechanism.