Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7

Abstract

Aims: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. Methods and Results: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single-and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87AE6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathioneSepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. Conclusions: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. Significance and Impact of the Study: The findings of this study provide important information on chitin degradation by microbial chitinases.

Bacterial chitinases and chitin-binding proteins as virulence factors

“…PKD domains have been suggested to directly mediate binding to chitin (Orikoshi et al, 2005). PKD domains may also promote protein-protein interactions, but the importance of this ability with regard to bacterial chitinases has not been investigated.…”

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

“…In chitinase A from the Gramnegative bacteria Alteromonas sp. strain O-7, a PKD domain has been shown to bind the cell wall polysaccharide chitin (51). The PDK domains of certain surface layer proteins have also been suggested to take part in the regulation of cell adhesion (52).…”

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

“…The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a ␤-helix fold and a conserved sequence area with two Trp residues in the hydrophobic core (2,3). However, the functional mechanism of the PKD domain in biopolymer hydrolases is largely unknown, except that the PKD domains in chitinase ChiA and collagenolytic serine protease deseasin MCP-01 are both reported to function as a binding domain (5,10). Genome sequence analysis and experimental results show that the PKD domain is common in the hydrolases of marine heterotrophic bacteria.…”

“…In addition to cell surface proteins, the PKD domain is found in many biopolymer hydrolases, such as chitinases (4,5), celluloses (6), and proteases (7)(8)(9), suggesting that it may play an important role in biopolymer degradation. The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a ␤-helix fold and a conserved sequence area with two Trp residues in the hydrophobic core (2,3).…”

“…For example, Gramella forsetii has 14 exported proteins with PKD domains (11). Chitin-binding protease AprIV, deseasin MCP-01, and many other marine deseasins contain one or two PKD domains (5,9). Therefore, elucidating the function and the mechanism of action of the PKD domains in these hydrolases would have universal importance, especially for clarifying the degradation mechanism of marine biopolymers.…”