title: Molecular cloning and characterization of a novel beta-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4.
abstract: We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kiyohara
authors.forename: Keishi
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Kuniko
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Araki
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002241:Carbohydrates / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity; D014733:Vibrio / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D014990:Xylans; D043325:Xylan Endo-1,3-beta-Xylosidase
references.citation: Appl Environ Microbiol. 2000 Apr;66(4):1741-3
references.pmid: 10742274
references.citation: Nature. 1960 Jul 2;187:82-3
references.pmid: 13852998
references.citation: J Biol Chem. 2001 Aug 17;276(33):31186-92
references.pmid: 11382747
references.citation: J Bacteriol. 2002 May;184(9):2399-403
references.pmid: 11948152
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Aug;60(Pt 8):1470-2
references.pmid: 15272180
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Agric Biol Chem. 1990 Apr;54(4):921-6
references.pmid: 1368551
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Phytochemistry. 1991;30(2):535-9
references.pmid: 1367268
references.citation: Anticancer Res. 1993 Nov-Dec;13(6A):2045-52
references.pmid: 8297113
references.citation: Mol Immunol. 1994 Mar;31(4):247-53
references.pmid: 7908118
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: Planta Med. 1995 Apr;61(2):120-5
references.pmid: 7753917
references.citation: Adv Microb Physiol. 1995;37:1-81
references.pmid: 8540419
references.citation: Caries Res. 1996;30(2):119-25
references.pmid: 8833135
references.citation: Biochemistry. 1996 Dec 17;35(50):16195-204
references.pmid: 8973192
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Curr Opin Microbiol. 1999 Jun;2(3):276-83
references.pmid: 10383869
references.citation: J Biol Chem. 1952 Mar;195(1):19-23
references.pmid: 14938350
references.citation: Lett Appl Microbiol. 2000 Jun;30(6):473-8
references.pmid: 10849279