title: Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1.
abstract: 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.
authors.affiliation: Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
authors.forename: Ce Mun
authors.initials: CM
authors.lastname: Tang
authors.forename: Mee-Len
authors.initials: ML
authors.lastname: Chye
authors.forename: Sathishkumar
authors.initials: S
authors.lastname: Ramalingam
authors.forename: Shi-Wen
authors.initials: SW
authors.lastname: Ouyang
authors.forename: Kai-Jun
authors.initials: KJ
authors.lastname: Zhao
authors.forename: Wimal
authors.initials: W
authors.lastname: Ubhayasekera
authors.forename: Sherry L
authors.initials: SL
authors.lastname: Mowbray
mesh_terms: D000371:Agglutination; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases