Bifunctional Family 3 Glycoside Hydrolases from Barley with α-l-Arabinofuranosidase and β-d-Xylosidase Activity

Abstract

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. 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. Heteroxylans are major constituents of cell walls in the Poaceae, which include many commercially important cereals and pasture grasses. In the endosperm of barley grains and in elongating coleoptiles, these polysaccharides may comprise 20–70% by weight of the walls (1Fincher G.B. Shewry P.R. Barley: Genetics, Molecular Biology and Bio/Technology. CAB Publishing, Wallingford, U. K.1992: 413-437Google Scholar) and consist of a backbone of (1 → 4)-β-linkedd-xylopyranosyl residues substituted predominantly with α-l-arabinofuranosyl residues. The α-l-arabinofuranosyl residues can be linked to O-3, O-2, or both O-3 and O-2 of xylanopyranosyl residues of the (1 → 4)-β-d-xylan backbone, and other substituents or short side chains are also detected in low abundance (2Viëtor R.J. Kormelink F.J.M. Angelino S.A.G.F. Voragen A.G.J. Carbohydr. Polym. 1994; 24: 113-118Crossref Scopus (35) Google Scholar, 3Carpita N.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 445-476Crossref PubMed Scopus (681) Google Scholar). The α-l-arabinofuranosyl residues can be esterified with hydroxycinnamic acids, in particular ferulic acid, which may form cross-bridges between adjacent arabinoxylan chains, or with lignin, by oxidative dimerization (4Iiyama K. Lam T.B.T. Stone B.A. Plant Physiol. 1993; 104: 315-320Crossref Scopus (639) Google Scholar).As observed with many wall components in higher plants, the arabinoxylans of primary cell walls can be restructured during normal growth and development. For example, newly synthesized arabinoxylans in maize coleoptiles are deposited in the walls in a highly substituted form, but arabinofuranosyl residues are removed later, and this leads to significant changes in the physicochemical properties of the polysaccharides and hence in the walls themselves (5Andrewartha K. Phillips D.R. Stone B.A. Carbohydr. Res. 1979; 77: 191-204Crossref Scopus (207) Google Scholar, 6Gibeaut D.M. Carpita N.C. Plant Physiol. 1991; 97: 551-561Crossref PubMed Scopus (187) Google Scholar). Removal of α-l-arabinofuranosyl residues is also observed when wall arabinoxylans are degraded (7Wilkie K.C.B. Adv. Carbohydr. Chem. Biochem. 1979; 36: 215-264Crossref Scopus (330) Google Scholar, 8Heyn A.N.J. Plant Sci. 1986; 45: 77-82Crossref Scopus (8) Google Scholar). The presence of α-l-arabinofuranosidases in germinated barley grain or in isolated aleurone layers has been taken as evidence that these enzymes perform this function during the mobilization of the starchy endosperm after cereal grain germination (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik G.B. Mol. PubMed Scopus Google this has also been to a of as arabinoxylan Biochem. PubMed Scopus Google Scholar, G.B. Biochem. PubMed Scopus Google α-l-arabinofuranosidases are designated can hydrolyze the glycoside 4′-nitrophenyl α-l-arabinofuranoside 4′-nitrophenyl 4′-nitrophenyl of XYL, 4′-nitrophenyl 4′-nitrophenyl of XYL, and are to be for changes in arabinoxylans during wall or this of enzymes may be Thus, α-l-arabinofuranosidases have been in glycoside and and of family and Voragen A.G.J. 1996; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, G.B. PubMed Scopus Google and Google Scholar). most of the enzymes are from or α-l-arabinofuranosidases have also been arabinoxylan which α-l-arabinofuranosyl residues from have been purified from germinated barley grain, and primary have been Biochem. PubMed Scopus Google Scholar, G.B. Biochem. PubMed Scopus Google Scholar). are other of the or of higher but in most amino acid is and is to the enzymes to about or to and have purified a bifunctional family 3 from young barley its and and its amino acid from The enzyme is to hydrolyze arabinoxylans a significant but an important in the of arabinoxylans through its to hydrolyze released from the polysaccharide by (1 → 4)-β-d-xylan In a family 3 purified and α-l-arabinofuranosidase and a β-d-xylosidase, both of which are family 3 glycoside Biochem. PubMed Scopus Google purified from extracts of germinated barley grain as in The purified enzymes have been designated ARA-I and XYL, respectively. α-l-arabinofuranosidase designated detected in the extracts but purified to The ARA-I and XYL enzymes have apparent molecular masses of 67 kDa and isoelectric of and 6.7, respectively. of and properties that α-l-arabinofuranosidase ARA-I can also hydrolyze and significant whereas the XYL enzyme has a or for Thus, the XYL enzyme hydrolyzes only but with a catalytic The catalytic efficiency for ARA-I of the of for and but slightly higher for For of have to the enzyme as an but ARA-I can hydrolyze both that be to as a bifunctional and that both be important for its family and α-l-arabinofuranosidases and in amino acid from the purified barley ARA-I and XYL cDNAs and amino acid that both enzymes have a that from in which are is a significant that the (1 → 4)-β-d-xylan in arabinoxylan in germinated barley grain is in the of aleurone layers but is in the and is to be released from aleurone layers only after cell Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google Scholar). In isolated aleurone α-l-arabinofuranosidases and are and can be detected in the than the (1 → G.B. Mol. PubMed Scopus Google Scholar). Thus, the from aleurone layers of and in arabinoxylan is the of ARA-I be with the cDNAs encode primary translation products of 748 amino acid residues The calculated molecular masses of the these amino acid are about is higher than the apparent molecular mass of 67 kDa observed during of the purified enzymes and a than other of the family 3 of glycoside G.B. PubMed Scopus Google Scholar). to the molecular masses of the enzymes and in both that the enzymes kDa in to for which the of for of the enzyme the predicted from the The amino acid also that the of the enzymes and that a COOH-terminal be this the weight of evidence that the of both ARA-I and XYL are to the of Thus, than 130 amino acid residues to have been removed from the during of the may be with the residues in the family 3 barley Stone B.A. G.B. Biol. Chem. 1996; PubMed Google Scholar). for COOH-terminal of the barley ARA-I and XYL enzymes can be this In the of the barley (1 → 4)-β-d-xylan both and COOH-terminal of the primary translation Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google of the amino acid of the mature enzymes with other of the family 3 glycoside that COOH-terminal in of the from other higher are in to those purified from the from Biochem. PubMed Scopus Google K. PubMed Google and Mol. PubMed Scopus Google Scholar) are than the barley enzymes and in to those predicted from observed for the family 3 ARA-I and XYL enzymes from barley can be in of predicted The of a family 3 from barley has been G.B. PubMed Scopus Google and is the only family 3 has been to of other family 3 enzymes G.B. PubMed Scopus Google Scholar). Molecular that the barley ARA-I and XYL enzymes have to that of the from the of the acid COOH-terminal of ARA-I and XYL, which is in the be barley has a for in and G.B. Plant PubMed Scopus Google Scholar, G.B. Carbohydr. Res. Scopus Google only residues of the the and the is between residues that G.B. Plant PubMed Scopus Google Scholar). The of the from the enzyme and is of and hence of G.B. PubMed Scopus Google Scholar). The barley ARA-I and XYL enzymes also in Both and can in catalytic a for this the of taken from the and the of the a observed about and in both and also is that the of ARA-I can both to the both enzymes hydrolyze but hydrolyzes substituted the that is removed from released by (1 → 4)-β-d-xylan that enzyme is to hydrolyze substituted or with to residues be to a of the in other family 3 enzymes G.B. PubMed Scopus Google Scholar, G.B. Plant PubMed Scopus Google and only be 3 glycoside from higher can be major amino acid G.B. Plant PubMed Scopus Google Scholar). the and the other and the ARA-I and XYL enzymes the the catalytic amino acid to and for ARA-I and and for XYL are in higher family 3 glycoside G.B. Plant PubMed Scopus Google Scholar) a for the in of the Thus, the amino acid in the that the of the is in the the have and the have a in the to of the the of the of family 3 enzymes is in the higher are from the is of in this In most the of enzymes by the genes in have been and the of is with a of The of the barley ARA-I has been for other of family 3 Biochem. PubMed Scopus Google Scholar). is that the barley ARA-I is from XYL in the and this may for of enzymes in this of family 3 acid of and with Res. 1994; PubMed Scopus Google are to The of the are the of the barley ARA-I and XYL of the genes with the of the enzymes and the presence of XYL mRNA in ARA-I mRNA to be or in low in the aleurone and of germinated grain is that α-l-arabinofuranosidase by has been in the isolated barley aleurone layers (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik G.B. Mol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the XYL to hydrolyze (1 → to but (1 → and In contrast, ARA-I (1 → to and (1 → to and enzyme arabinoxylan a significant but both ARA-I and XYL rapidly released from or that released from the arabinoxylan by the of (1 → 4)-β-d-xylan endohydrolase. The low of in these 3 that this polysaccharide about α-l-arabinofuranosyl residues G.B. Stone B.A. Plant Physiol. Google but that enzyme can substituted residues in In be that the ARA-I and XYL enzymes in hydrolysis of released from arabinoxylans by in germinated barley The enzymes also an important during cell wall in elongating coleoptiles and in other during normal growth and development. Heteroxylans are major constituents of cell walls in the Poaceae, which include many commercially important cereals and pasture grasses. In the endosperm of barley grains and in elongating coleoptiles, these polysaccharides may comprise 20–70% by weight of the walls (1Fincher G.B. Shewry P.R. Barley: Genetics, Molecular Biology and Bio/Technology. CAB Publishing, Wallingford, U. K.1992: 413-437Google Scholar) and consist of a backbone of (1 → 4)-β-linkedd-xylopyranosyl residues substituted predominantly with α-l-arabinofuranosyl residues. The α-l-arabinofuranosyl residues can be linked to O-3, O-2, or both O-3 and O-2 of xylanopyranosyl residues of the (1 → 4)-β-d-xylan backbone, and other substituents or short side chains are also detected in low abundance (2Viëtor R.J. Kormelink F.J.M. Angelino S.A.G.F. Voragen A.G.J. Carbohydr. Polym. 1994; 24: 113-118Crossref Scopus (35) Google Scholar, 3Carpita N.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 445-476Crossref PubMed Scopus (681) Google Scholar). The α-l-arabinofuranosyl residues can be esterified with hydroxycinnamic acids, in particular ferulic acid, which may form cross-bridges between adjacent arabinoxylan chains, or with lignin, by oxidative dimerization (4Iiyama K. Lam T.B.T. Stone B.A. Plant Physiol. 1993; 104: 315-320Crossref Scopus (639) Google Scholar). observed with many wall components in higher plants, the arabinoxylans of primary cell walls can be restructured during normal growth and development. For example, newly synthesized arabinoxylans in maize coleoptiles are deposited in the walls in a highly substituted form, but arabinofuranosyl residues are removed later, and this leads to significant changes in the physicochemical properties of the polysaccharides and hence in the walls themselves (5Andrewartha K. Phillips D.R. Stone B.A. Carbohydr. Res. 1979; 77: 191-204Crossref Scopus (207) Google Scholar, 6Gibeaut D.M. Carpita N.C. Plant Physiol. 1991; 97: 551-561Crossref PubMed Scopus (187) Google Scholar). Removal of α-l-arabinofuranosyl residues is also observed when wall arabinoxylans are degraded (7Wilkie K.C.B. Adv. Carbohydr. Chem. Biochem. 1979; 36: 215-264Crossref Scopus (330) Google Scholar, 8Heyn A.N.J. Plant Sci. 1986; 45: 77-82Crossref Scopus (8) Google Scholar). The presence of α-l-arabinofuranosidases in germinated barley grain or in isolated aleurone layers has been taken as evidence that these enzymes perform this function during the mobilization of the starchy endosperm after cereal grain germination (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik G.B. Mol. PubMed Scopus Google this has also been to a of as arabinoxylan Biochem. PubMed Scopus Google Scholar, G.B. Biochem. PubMed Scopus Google Scholar). α-l-arabinofuranosidases are designated can hydrolyze the glycoside 4′-nitrophenyl α-l-arabinofuranoside 4′-nitrophenyl 4′-nitrophenyl of XYL, 4′-nitrophenyl 4′-nitrophenyl of XYL, and are to be for changes in arabinoxylans during wall or this of enzymes may be Thus, α-l-arabinofuranosidases have been in glycoside and and of family and Voragen A.G.J. 1996; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, G.B. PubMed Scopus Google and Google Scholar). most of the enzymes are from or α-l-arabinofuranosidases have also been arabinoxylan which α-l-arabinofuranosyl residues from have been purified from germinated barley grain, and primary have been Biochem. PubMed Scopus Google Scholar, G.B. Biochem. PubMed Scopus Google Scholar). are other of the or of higher but in most amino acid is and is to the enzymes to about or to and have purified a bifunctional family 3 from young barley its and and its amino acid from The enzyme is to hydrolyze arabinoxylans a significant but an important in the of arabinoxylans through its to hydrolyze released from the polysaccharide by (1 → 4)-β-d-xylan In a family 3 purified and α-l-arabinofuranosidase and a β-d-xylosidase, both of which are family 3 glycoside Biochem. PubMed Scopus Google purified from extracts of germinated barley grain as in The purified enzymes have been designated ARA-I and XYL, respectively. α-l-arabinofuranosidase designated detected in the extracts but purified to The ARA-I and XYL enzymes have apparent molecular masses of 67 kDa and isoelectric of and 6.7, respectively. of and properties that α-l-arabinofuranosidase ARA-I can also hydrolyze and significant whereas the XYL enzyme has a or for Thus, the XYL enzyme hydrolyzes only but with a catalytic The catalytic efficiency for ARA-I of the of for and but slightly higher for For of have to the enzyme as an but ARA-I can hydrolyze both that be to as a bifunctional and that both be important for its family and α-l-arabinofuranosidases and in amino acid from the purified barley ARA-I and XYL cDNAs and amino acid that both enzymes have a that from in which are is a significant that the (1 → 4)-β-d-xylan in arabinoxylan in germinated barley grain is in the of aleurone layers but is in the and is to be released from aleurone layers only after cell Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google Scholar). In isolated aleurone α-l-arabinofuranosidases and are and can be detected in the than the (1 → G.B. Mol. PubMed Scopus Google Scholar). Thus, the from aleurone layers of and in arabinoxylan is the of ARA-I be with the cDNAs encode primary translation products of 748 amino acid residues The calculated molecular masses of the these amino acid are about is higher than the apparent molecular mass of 67 kDa observed during of the purified enzymes and a than other of the family 3 of glycoside G.B. PubMed Scopus Google Scholar). to the molecular masses of the enzymes and in both that the enzymes kDa in to for which the of for of the enzyme the predicted from the The amino acid also that the of the enzymes and that a COOH-terminal be this the weight of evidence that the of both ARA-I and XYL are to the of Thus, than 130 amino acid residues to have been removed from the during of the may be with the residues in the family 3 barley Stone B.A. G.B. Biol. Chem. 1996; PubMed Google Scholar). for COOH-terminal of the barley ARA-I and XYL enzymes can be this In the of the barley (1 → 4)-β-d-xylan both and COOH-terminal of the primary translation Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google of the amino acid of the mature enzymes with other of the family 3 glycoside that COOH-terminal in of the from other higher are in to those purified from the from Biochem. PubMed Scopus Google K. PubMed Google and Mol. PubMed Scopus Google Scholar) are than the barley enzymes and in to those predicted from observed for the family 3 ARA-I and XYL enzymes from barley can be in of predicted The of a family 3 from barley has been G.B. PubMed Scopus Google and is the only family 3 has been to of other family 3 enzymes G.B. PubMed Scopus Google Scholar). Molecular that the barley ARA-I and XYL enzymes have to that of the from the of the acid COOH-terminal of ARA-I and XYL, which is in the be barley has a for in and G.B. Plant PubMed Scopus Google Scholar, G.B. Carbohydr. Res. Scopus Google only residues of the the and the is between residues that G.B. Plant PubMed Scopus Google Scholar). The of the from the enzyme and is of and hence of G.B. PubMed Scopus Google Scholar). The barley ARA-I and XYL enzymes also in Both and can in catalytic a for this the of taken from the and the of the a observed about and in both and also is that the of ARA-I can both to the both enzymes hydrolyze but hydrolyzes substituted the that is removed from released by (1 → 4)-β-d-xylan that enzyme is to hydrolyze substituted or with to residues be to a of the in other family 3 enzymes G.B. PubMed Scopus Google Scholar, G.B. Plant PubMed Scopus Google and only be 3 glycoside from higher can be major amino acid G.B. Plant PubMed Scopus Google Scholar). the and the other and the ARA-I and XYL enzymes the the catalytic amino acid to and for ARA-I and and for XYL are in higher family 3 glycoside G.B. Plant PubMed Scopus Google Scholar) a for the in of the Thus, the amino acid in the that the of the is in the the have and the have a in the to of the the of the of family 3 enzymes is in the higher are from the is of in this In most the of enzymes by the genes in have been and the of is with a of The of the barley ARA-I has been for other of family 3 Biochem. PubMed Scopus Google Scholar). is that the barley ARA-I is from XYL in the and this may for of enzymes in this the of the barley ARA-I and XYL of the genes with the of the enzymes and the presence of XYL mRNA in ARA-I mRNA to be or in low in the aleurone and of germinated grain is that α-l-arabinofuranosidase by has been in the isolated barley aleurone layers (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik G.B. Mol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the XYL to hydrolyze (1 → to but (1 → and In contrast, ARA-I (1 → to and (1 → to and enzyme arabinoxylan a significant but both ARA-I and XYL rapidly released from or that released from the arabinoxylan by the of (1 → 4)-β-d-xylan endohydrolase. The low of in these 3 that this polysaccharide about α-l-arabinofuranosyl residues G.B. Stone B.A. Plant Physiol. Google but that enzyme can substituted residues in In be that the ARA-I and XYL enzymes in hydrolysis of released from arabinoxylans by in germinated barley The enzymes also an important during cell wall in elongating coleoptiles and in other during normal growth and development. An α-l-arabinofuranosidase and a β-d-xylosidase, both of which are family 3 glycoside Biochem. PubMed Scopus Google purified from extracts of germinated barley grain as in The purified enzymes have been designated ARA-I and XYL, respectively. α-l-arabinofuranosidase designated detected in the extracts but purified to The ARA-I and XYL enzymes have apparent molecular masses of 67 kDa and isoelectric of and 6.7, respectively. of and properties that α-l-arabinofuranosidase ARA-I can also hydrolyze and significant whereas the XYL enzyme has a or for Thus, the XYL enzyme hydrolyzes only but with a catalytic The catalytic efficiency for ARA-I of the of for and but slightly higher for For of have to the enzyme as an but ARA-I can hydrolyze both that be to as a bifunctional and that both be important for its family and α-l-arabinofuranosidases and in amino acid from the purified barley ARA-I and XYL cDNAs and amino acid that both enzymes have a that from in which are is a significant that the (1 → 4)-β-d-xylan in arabinoxylan in germinated barley grain is in the of aleurone layers but is in the and is to be released from aleurone layers only after cell Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google Scholar). In isolated aleurone α-l-arabinofuranosidases and are and can be detected in the than the (1 → G.B. Mol. PubMed Scopus Google Scholar). Thus, the from aleurone layers of and in arabinoxylan is the of ARA-I be with the cDNAs encode primary translation products of 748 amino acid residues The calculated molecular masses of the these amino acid are about is higher than the apparent molecular mass of 67 kDa observed during of the purified enzymes and a than other of the family 3 of glycoside G.B. PubMed Scopus Google Scholar). to the molecular masses of the enzymes and in both that the enzymes kDa in to for which the of for of the enzyme the predicted from the The amino acid also that the of the enzymes and that a COOH-terminal be this the weight of evidence that the of both ARA-I and XYL are to the of Thus, than 130 amino acid residues to have been removed from the during of the may be with the residues in the family 3 barley Stone B.A. G.B. Biol. Chem. 1996; PubMed Google Scholar). for COOH-terminal of the barley ARA-I and XYL enzymes can be this In the of the barley (1 → 4)-β-d-xylan both and COOH-terminal of the primary translation Plant 191-204Crossref PubMed Scopus Google Scholar, G.B. Sci. Scopus Google Scholar). of the amino acid of the mature enzymes with other of the family 3 glycoside that COOH-terminal in of the from other higher are in to those purified from the from Biochem. PubMed Scopus Google K. PubMed Google and Mol. PubMed Scopus Google Scholar) are than the barley enzymes and in to those predicted from The observed for the family 3 ARA-I and XYL enzymes from barley can be in of predicted The of a family 3 from barley has been G.B. PubMed Scopus Google and is the only family 3 has been to of other family 3 enzymes G.B. PubMed Scopus Google Scholar). Molecular that the barley ARA-I and XYL enzymes have to that of the from the of the acid COOH-terminal of ARA-I and XYL, which is in the be The barley has a for in and G.B. Plant PubMed Scopus Google Scholar, G.B. Carbohydr. Res. Scopus Google only residues of the the and the is between residues that G.B. Plant PubMed Scopus Google Scholar). The of the from the enzyme and is of and hence of G.B. PubMed Scopus Google Scholar). The barley ARA-I and XYL enzymes also in Both and can in catalytic a for this the of taken from the and the of the a observed about and in both and also is that the of ARA-I can both In to the both enzymes hydrolyze but hydrolyzes substituted the that is removed from released by (1 → 4)-β-d-xylan that enzyme is to hydrolyze substituted or with to residues be to a of the in other family 3 enzymes G.B. PubMed Scopus Google Scholar, G.B. Plant PubMed Scopus Google and only be 3 glycoside from higher can be major amino acid G.B. Plant PubMed Scopus Google Scholar). the and the other and the ARA-I and XYL enzymes the the catalytic amino acid to and for ARA-I and and for XYL are in higher family 3 glycoside G.B. Plant PubMed Scopus Google Scholar) a for the in of the Thus, the amino acid in the that the of the is in the the have and the have a in the to of the the of the of family 3 enzymes is in the higher are from the is of in this In most the of enzymes by the genes in have been and the of is with a of The of the barley ARA-I has been for other of family 3 Biochem. PubMed Scopus Google Scholar). is that the barley ARA-I is from XYL in the and this may for of enzymes in this the of the barley ARA-I and XYL of the genes with the of the enzymes and the presence of XYL mRNA in ARA-I mRNA to be or in low in the aleurone and of germinated grain is that α-l-arabinofuranosidase by has been in the isolated barley aleurone layers (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik G.B. Mol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the XYL to hydrolyze (1 → to but (1 → and In contrast, ARA-I (1 → to and (1 → to and enzyme arabinoxylan a significant but both ARA-I and XYL rapidly released from or that released from the arabinoxylan by the of (1 → 4)-β-d-xylan endohydrolase. The low of in these 3 that this polysaccharide about α-l-arabinofuranosyl residues G.B. Stone B.A. Plant Physiol. Google but that enzyme can substituted residues in In be that the ARA-I and XYL enzymes in hydrolysis of released from arabinoxylans by in germinated barley The enzymes also an important during cell wall in elongating coleoptiles and in other during normal growth and development. for the aleurone for with the for with the and and for of the mass and in of the