A New Putative Chitinase from Reticulitermes speratus KMT001

Abstract

Termites are pests that cause serious economic and cultural damage by digesting wood cellulose. Termites are arthropods and have an epidermis surrounded by a chitin layer. To maintain a healthy epidermis, termites have chitinase (${\beta}$-1,4-poly-N-acetyl glucosamidinase, EC 3.2.1.14), an enzyme that hydrolyzes the ${\beta}$-1,4 bond of chitin. In this study, the amino acid sequence of the gene, which is presumed to be termite chitinolytic enzyme (NCBI accession no. KC477099), was obtained from a transcriptomic analysis of Reticulitermes speratus KMT001 in Bukhan Mountain, Korea. An NCBI protein BLAST search confirmed that the protein is a glycoside hydrolase family 18 (GH18). The highest homology value found was 47%, with a chitinase from Araneus ventricosus. Phylogenetic analysis indicated that the KC477099 protein has the same origins as those of arthropods but has a very low similarity with other arthropod chitinases, resulting in separation at an early stage of evolution. The KC477099 protein contains two conserved motifs, which encode the general enzymatic characteristics of the GH18 group. The amino acid sequences $Asp^{156}-Trp^{157}-Glu^{158}$, which play an important role in the enzymatic activity of the GH18 group, were also present. This study suggests that the termite KC477099 protein is a new type of chitinase, which is evolutionarily distant from other insect chitinases.

Fig. 1. Multiple amino acid sequence alignment of KC477099 from R. speratus KMT001 and chitinase from 19 strains obtained from a protein BLAST search of the National Center for Biotechnology Information. Motifs I and II are indicated on the top of the alignments. The bold characteristics indicate the consensus sequences in the active sites of GH18. The star marks on the bottom of the alignments indicate the active sites of GH18 chitolectin chitotriosidase.

Fig. 2. Phylogenetic analysis of KC477099 from R. speratus KMT001 and chitinase from 19 strains from a protein BLAST search at the National Center for Biotechnology Information. A neighbor-joining method with a bootstrap of 1,000 replications was used. The order and phylum of each strain are listed on the right side of the figure. KC477099 is indicated using a black arrow. Saccoglossus kowalevskii class is listed because it has not been assigned to an order.

Table 1. Putative chitinase genes from transcriptomic analysis of R. speratus KMT001.

Table 2. Results of TBLASTN searches using the amino acid sequence of KC477099 (contig00176) ORF from R. speratus KMT001.