scholarly journals 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

2006 ◽  
Vol 399 (1) ◽  
pp. 77-90 ◽  
Author(s):  
Maria Hrmova ◽  
Rachel A. Burton ◽  
Peter Biely ◽  
Jelle Lahnstein ◽  
Geoffrey B. Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Three Dimensional ◽  
Complete Sequence ◽  
Degree Of Polymerization ◽  
Size Exclusion ◽  
Basal Region ◽  
Three Dimensional Model ◽  
Hordeum Vulgare L ◽  
Ph Optimum ◽  
Hydrolysis Of

A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-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 Km constants of 0.16 mg·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)-β-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)-β-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)-β-D-manno-oligosaccharides to Man is a family GH1 β-D-mannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134–11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase.

scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones

2001 ◽  
Vol 356 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Robert C. LEE ◽  
Rachel A. BURTON ◽  
Maria HRMOVA ◽  
Geoffrey B. FINCHER
Keyword(s):  
Catalytic Efficiency ◽  
Exchange Chromatography ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Hordeum Vulgare L ◽  
Ph Optimum ◽  
Catalytic Rate Constant ◽  
Arabinoxylan Arabinofuranohydrolase ◽  
Mature Enzyme ◽  
Hydrolysis Of

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH4)2SO4 and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65kDa and releases l-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (kcat) of 6.9s−1 and a catalytic efficiency factor (kcat/Km) of 0.76 (ml·s−1·mg−1). Whereas the hydrolysis of α-l-arabinofuranosyl residues linked to C(O)3 of backbone (1 → 4)-β-xylosyl residues proceeds at the fastest rate, α-l-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.

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

2006 ◽  
Vol 394 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Qisen Zhang ◽  
Maria Hrmova ◽  
Neil J. Shirley ◽  
Jelle Lahnstein ◽  
Geoffrey B. Fincher
Keyword(s):  
Aqueous Ethanol ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Biological Significance ◽  
Mrna Levels ◽  
Root Tips ◽  
Three Dimensional Model ◽  
Cell Wall Polysaccharides ◽  
Hordeum Vulgare L ◽  
Nucleotide Sugars

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)-β-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 Km values were 40 μM for UDP-Gal and 55 μM 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.

Ultrastructure and Senescence in an Achloroplastic Mutant of Hordeum vulgare L. cv. Dyan

1992 ◽  
Vol 50 (1) ◽  
pp. 844-845
Author(s):  
R.H.M. Cross ◽  
C.E.J. Botha ◽  
A.K. Cowan ◽  
B.J. Hartley
Keyword(s):  
Cell Wall ◽  
Hordeum Vulgare ◽  
Leaf Tissue ◽  
Ultrastructural Changes ◽  
Hordeum Vulgare L ◽  
Mesophyll Cells ◽  
Lethal Mutant ◽  
Cytoplasmic Matrix ◽  
Close Proximity ◽  
Pinocytotic Vesicles

Senescence is an ordered degenerative process leading to death of individual cells, organs and organisms. The detection of a conditional lethal mutant (achloroplastic) of Hordeum vulgare has enabled us to investigate ultrastructural changes occurring in leaf tissue during foliar senescence.Examination of the tonoplast structure in six and 14 day-old mutant tissue revealed a progressive degeneration and disappearance of the membrane, apparently starting by day six in the vicinity of the mitochondria associated with the degenerating proplastid (Fig. 1.) where neither of the plastid membrane leaflets is evident (arrows, Fig. 1.). At this stage there was evidence that the mitochondrial membranes were undergoing retrogressive changes, coupled with disorganization of cristae (Fig. 2.). Proplastids (P) lack definitive prolamellar bodies. The cytoplasmic matrix is largely agranular, with few endoplasmic reticulum (ER) cisternae or polyribosomal aggregates. Interestingly, large numbers of actively-budding dictysomes, associated with pinocytotic vesicles, were observed in close proximity to the plasmalemma of mesophyll cells (Fig. 3.). By day 14 however, mesophyll cells showed almost complete breakdown of subcellular organelle structure (Fig. 4.), and further evidence for the breakdown of the tonoplast. The final stage of senescence is characterized by the solubilization of the cell wall due to expression and activity of polygalacturonase and/or cellulose. The presence of dictyosomes with associated pinocytotic vesicles formed from the mature face, in close proximity to both the plasmalemma and the cell wall, would appear to support the model proposed by Christopherson for the secretion of cellulase. This pathway of synthesis is typical for secretory glycoproteins.

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