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.
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.
Kiyohara
Masashi
M
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Sakaguchi
Keishi
K
Yamaguchi
Kuniko
K
Araki
Toshiyoshi
T
Nakamura
Takashi
T
Ito
Makoto
M
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
D002241:Carbohydrates; D014990:Xylans; D043325:Xylan Endo-1,3-beta-Xylosidase
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