Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

Abstract

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

Disturbed processing of the carbohydrate‐binding module of family 54 significantly impairs its binding to polysaccharides

“…Such a cleavage was demonstrated for rtCBM54 as a module in both native and recombinant Lic16A as well as for an individual module expressed in Escherichia coli . A similar cleavage was revealed in the full‐length chitinase ChiW from Paenibacillus expressed in E. coli as demonstrated by the analysis of its 3D structure . The cleavage of the polypeptide chain does not result in dissociation of two parts of CBM54 .…”

“…Recently, the 3D structure of the multimodular chitinase ChiW bearing a CBM54 has been determined. Structurally, the CBM54 of ChiW is a right‐handed β‐helix similar to that of polysaccharide lyases . No clefts typical of type B CBMs or pockets typical of type C ones have been revealed.…”

The deduced role of a chitinase containing two nonsynergistic catalytic domains

“…The crystal structures of chitinases with one single GH18 domain have been extensively studied in archaea (Tsuji et al, 2010), bacteria (Perrakis et al, 1994;van Aalten et al, 2000;Songsiriritthigul et al, 2008;Hsieh et al, 2010;Busby et al, 2012;Payne et al, 2012;Madhuprakash et al, 2013;Malecki et al, 2013;Ü stok et al, 2015;Itoh et al, 2016), fungi (Hollis et al, 2000;Rao et al, 2005;Hurtado-Guerrero & van Aalten, 2007;Schü ttelkopf et al, 2010;Yang et al, 2010), plants (Terwisscha van Scheltinga et al, 1994;Cavada et al, 2006;Ohnuma, Numata, Osawa, Mizuhara, Lampela et al, 2011;Ohnuma, Numata, Osawa, Mizuhara, Vå rum et al, 2011;Kitaoku et al, 2015;Masuda et al, 2015;Umemoto et al, 2015), insects Liu et al, 2017) and humans (Fusetti et al, 2002;Olland et al, 2009). Although the overall structure of these GH18 domains is a (/) 8 -barrel with a substrate-binding cleft on the top side, they can be differentiated by the shapes of the substrate-binding clefts and the presence or absence of insertion domains.…”

“…Chitinases with two GH18 domains have been discovered in viruses, archaea, bacteria and insects (Hiramatsu et al, 1999;Tanaka et al, 2001;Howard et al, 2004;Arakane & Muthukrishnan, 2010;Itoh et al, 2016). In some of these chitinases, the two catalytic domains work synergistically because the two domains in combination exhibit a significantly higher activity than the sum of their individual activities.…”

“…A different mechanism is found for ChiW, a chitinase from Paenibacillus sp. FPU-7 (Itoh et al, 2016). Structural studies indicate that the two GH18 domains are assembled into a compact catalytic region together with two immunoglobulin-like fold domains.…”