The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/ß)8 TIM barrel catalytic domain; and iii) the α+ß insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBDVhChiA. The recombinant ChBDVhChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBDVhChiA contained ß-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBDVhChiA bound to both α- and ß-chitins, with the greatest affinity for ß-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions.

Azide anions inhibit GH-18 endochitinase and GH-20 Exo ß-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin as its sole source of energy. In the course of chitin degradation, the bacterium primarily secretes an endochitinase A (VhChiA) to hydrolyze chitin, generating chitooligosaccharide fragments that are readily transported into the cell and broken down to GlcNAc monomers by an exo ß-N-acetylglucosaminidase (VhGlcNAcase). Here we report that sodium salts, especially sodium azide, inhibit two classes of these chitin-degrading enzymes (VhChiA and VhGlcNAcase) with distinct modes of action. Kinetic analysis of the enzymatic hydrolysis of pNP-glycoside substrates reveals that sodium azide inhibition of VhChiA has a mixed-type mode, but that it inhibits VhGlcNAcase competitively. We propose that azide anions inhibit chitinase activity by acting as strong nucleophiles that attack Cγ of the catalytic Glu or Cß of the neighbouring Asp residues. Azide anions may bind not only to the catalytic centre, but also to the other subsites in the substrate-binding cleft of VhChiA. In contrast, azide anions may merely occupy the small-binding pocket of VhGlcNAcase, thereby blocking the accessibility of its active site by short-chain substrates.

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase.

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc(n), with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc(n). On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.