Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

文献类型： 外文期刊

第一作者： Tang, CM

作者： Tang, CM;Chye, ML;Ramalingam, S;Ouyang, SW;Zhao, KJ;Ubhayasekera, W;Mowbray, SL

作者机构：

关键词： agglutination;glycoside hydrolases;homology modeling;lectin;pathogenesis-related protein;site-directed mutagenesis

期刊名称：PLANT MOLECULAR BIOLOGY （ 影响因子：4.076； 五年影响因子：4.89 ）

ISSN： 0167-4412

年卷期： 2004 年 56 卷 2 期

页码：

收录情况： SCI

摘要： We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2 showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using alpha-mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by alpha-mannosidase. BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity. H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis. E234A showed 36% retention of activity and substitution Y269D, 50%. The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.

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