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. 2001 May 15;356(Pt 1):181–189. doi: 10.1042/0264-6021:3560181

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

R C Lee 1, R A Burton 1, M Hrmova 1, G B Fincher 1
PMCID: PMC1221826  PMID: 11336650

Abstract

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 (NH(4))(2)SO(4) and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65 kDa and releases L-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (k(cat)) of 6.9 s(-1) and a catalytic efficiency factor (k(cat)/K(m)) of 0.76 (ml x s(-1) x mg(-1)). Whereas the hydrolysis of alpha-L-arabinofuranosyl residues linked to C(O)3 of backbone (1-->4)-beta-xylosyl residues proceeds at the fastest rate, alpha-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.

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Selected References

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  1. Banik M., Li C. D., Langridge P., Fincher G. B. Structure, hormonal regulation, and chromosomal location of genes encoding barley (1-->4)-beta-xylan endohydrolases. Mol Gen Genet. 1997 Feb 20;253(5):599–608. doi: 10.1007/s004380050362. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Carpita Nicholas C. STRUCTURE AND BIOGENESIS OF THE CELL WALLS OF GRASSES. Annu Rev Plant Physiol Plant Mol Biol. 1996 Jun;47(NaN):445–476. doi: 10.1146/annurev.arplant.47.1.445. [DOI] [PubMed] [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ferré H., Broberg A., Duus J. O., Thomsen K. K. A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley analysis of substrate preference and specificity by nano-probe NMR. Eur J Biochem. 2000 Nov;267(22):6633–6641. doi: 10.1046/j.1432-1327.2000.01758.x. [DOI] [PubMed] [Google Scholar]
  6. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gibeaut D. M., Carpita N. C. Tracing cell wall biogenesis in intact cells and plants : selective turnover and alteration of soluble and cell wall polysaccharides in grasses. Plant Physiol. 1991 Oct;97(2):551–561. doi: 10.1104/pp.97.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henrissat B. Glycosidase families. Biochem Soc Trans. 1998 May;26(2):153–156. doi: 10.1042/bst0260153. [DOI] [PubMed] [Google Scholar]
  9. Hrmova M., Fincher G. B. Purification and properties of three (1-->3)-beta-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare). Biochem J. 1993 Jan 15;289(Pt 2):453–461. doi: 10.1042/bj2890453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hrmova M., Harvey A. J., Wang J., Shirley N. J., Jones G. P., Stone B. A., Høj P. B., Fincher G. B. Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone. J Biol Chem. 1996 Mar 1;271(9):5277–5286. doi: 10.1074/jbc.271.9.5277. [DOI] [PubMed] [Google Scholar]
  11. Kokubo A., Kuraishi S., Sakurai N. Culm strength of barley : correlation among maximum bending stress, cell wall dimensions, and cellulose content. Plant Physiol. 1989 Nov;91(3):876–882. doi: 10.1104/pp.91.3.876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Matsuo N., Kaneko S., Kuno A., Kobayashi H., Kusakabe I. Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901. Biochem J. 2000 Feb 15;346(Pt 1):9–15. [PMC free article] [PubMed] [Google Scholar]
  14. SMOGYI M. Notes on sugar determination. J Biol Chem. 1952 Mar;195(1):19–23. [PubMed] [Google Scholar]
  15. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Taiz L., Honigman W. A. Production of cell wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic Acid. Plant Physiol. 1976 Sep;58(3):380–386. doi: 10.1104/pp.58.3.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Whitehead T. R. Nucleotide sequences of xylan-inducible xylanase and xylosidase/arabinosidase genes from Bacteroides ovatus V975. Biochim Biophys Acta. 1995 May 11;1244(1):239–241. doi: 10.1016/0304-4165(95)00051-c. [DOI] [PubMed] [Google Scholar]
  18. Zverlov V. V., Liebl W., Bachleitner M., Schwarz W. H. Nucleotide sequence of arfB of Clostridium stercorarium, and prediction of catalytic residues of alpha-L-arabinofuranosidases based on local similarity with several families of glycosyl hydrolases. FEMS Microbiol Lett. 1998 Jul 15;164(2):337–343. doi: 10.1111/j.1574-6968.1998.tb13107.x. [DOI] [PubMed] [Google Scholar]

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