The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity
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Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.
KeywordsChitinase Characterization Catalytic domain Insertion region Listeria species C-terminal truncation
We are grateful to Kazuaki Okawa and Daiya Kuribara for their valuable suggestions and technical assistance.
This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number 17K07729); the Strategic Research Foundation Grant-aided Project for Private Universities of the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT) (Grant Number S1411005); the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan; and the Project Research Grant from the Research Institute of Science and Technology, Kogakuin University.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals.
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