A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

Abstract

The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the ␤-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.Chitinases (EC 3.2.1.14) are glycosyl hydrolases which catalyze the degradation of chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine. Recently, chitinases have been receiving renewed attention because of their possible application for the biological control of chitin-containing organisms and also for the exploitation of natural chitinous materials. They are present in a wide range of organisms including bacteria, insects, viruses, plants, and animals and play important physiological and ecological roles. Chitinases so far sequenced are classified in two different families (namely, families 18 and 19) of the classification of glycosyl hydrolases based on amino acid sequence similarities (13,14). Family 18 contains chitinases from bacteria, fungi, viruses, and animals and class III and V chitinases from plants. On the other hand, class I, II, and IV plant chitinases belong to family 19, and this family solely comprises chitinases of plant origin. The two different families of chitinases display no sequence similarities with each other and have different three-dimensional structures (5).Oligosaccharides produced from partially acetylated chitosan by microbial chitinases have been previously studied in an attempt to clarify their specificity for ␤-1,4-N-acetylglucosaminic versus ␤-1,4-glucosaminic linkages (26, 27, 30). Except for chitinase C-1 from Streptomyces griseus HUT 6037, all of the examined microbial chitinases hydrolyzed GlcNAc-GlcNA...

Functional Analysis of the Chitin-binding Domain of a Family 19 Chitinase fromStreptomyces griseusHUT6037: Substrate-binding Affinity andcis-Dominant Increase of Antifungal Function

“…The mixtures were incubated for 3 (ChiC) or 24 h (CatDChiC and ChBDChiC) on ice with occasional mixing, then centrifuged at 15,000×g for 20 min at 49 C the protein concentration in the supernatant containing free proteins was measured. The amount of bound proteins was estimated by subtract- Numbers indicate amino acid positions relative to theˆrst methionine of ChiC (9). Coding regions of relevant domains were inserted in-frame into the expression vector pET-22b(+) using restriction sites created in PCR-ampliˆed fragments (see Materials and Methods).…”

“…8) Chitinase C (ChiC) from S. griseus HUT6037 was theˆrst family 19 chitinase discovered in an organism other than higher plants. 9) The bacterial family 19 chitinases were then identiˆed in other bacteria including Burkholderia gladioli, Vibrio cholerae, Haemophilus in‰uenzae, and Pseudomonas aeruginosa, [10][11][12][13] as well as in other Streptomyces species. 14) Several genes for family 19 chitinases have been identiˆed in the Caenorhabditis elegans genome.…”

Transcriptional co-regulation of five chitinase genes scattered on the Streptomyces coelicolor A3(2) chromosome

“…Family 19 chitinases also differ from family 18 chitinases in their catalytic mechanisms (Armand et al, 1994 ;Iseli et al, 1996). Streptomyces are unique micro-organisms in possessing family 19 chitinase genes, which are usually found only in higher plants (Ohno et al, 1996 ;Saito et al, 1999 ;.…”

“…Streptomyces are saprophytic soil bacteria known as major decomposers of chitin in soil. The genes encoding chitinases (EC 3;2;1;14) -enzymes that hydrolyse chitin -have been cloned from them (Robbins et al, 1988 ;Miyashita et al, 1991Miyashita et al, , 1997Blaak et al, 1993 ;Tsujibo et al, 1993Tsujibo et al, , 2000Ohno et al, 1996 ;Saito et al, 1999). The products of these Streptomyces chitinase genes are classified, based on amino acid sequences analysis, into two distinct families of glycosyl hydrolases, families 18 and 19 (Henrissat, 1991 ;Henrissat & Bairoch, 1993).…”

Insight Into Genes Involved in the Production of Extracellular Chitinase in a Biocontrol Bacterium Lysobacter enzymogenes C-3

“…Chitinases are divided into two different families (18 and 19) based on amino acid sequence similarities (Henrissat and Davies, 1997). Plant chitinases and some bacterial chitinases from Streptomyces species are grouped in the family of 19 chitinases which are known to have strong antifungal activities (Cohen-Kupiec and Chet, 1998;Ohno et al, 1996). Most bacterial chitinases, including most of the Gram negative bacteria and Bacillus species belong to family 18.…”