Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops.

Brassica juncea chitinase is an endo-acting, pathogenesis-related protein that is classified into glycoside hydrolase family 19, with highest homology (50-60%) in its catalytic domain to class I plant chitinases. Here we report X-ray structures of the chitinase catalytic domain from wild-type (apo, as well as with chloride ions bound) and a Glu234Ala mutant enzyme, solved by molecular replacement and refined at 1.53, 1.8 and 1.7 A resolution, respectively. Confirming our earlier mutagenesis studies, the active-site residues are identified as Glu212 and Glu234. Glu212 is believed to be the catalytic acid in the reaction, whereas Glu234 is thought to have a dual role, both activating a water molecule in its attack on the anomeric carbon, and stabilizing the charged intermediate. The molecules in the various structures differ significantly in the conformation of a number of loops that border the active-site cleft. The differences suggest an opening and closing of the enzyme during the catalytic cycle. Chitin is expected to dock first near Glu212, which will protonate it. Conformational changes then bring Glu234 closer, allowing it to assist in the following steps. These observations provide important insights into catalysis in family 19 chitinases.

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

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.