A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 °C, with an optimum pH of pH 7–7.5. The activity was enhanced by divalent cations, such as Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 Å, which possesses a β-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the pK_a of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and β-helix polysaccharide lyases.

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.

The α‐1,3‐glucanase from Paenibacillus glycanilyticus FH11 (Agl‐FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α‐1,3‐glucan with an endo‐action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α‐1,3‐tetraglucan into glucose and α‐1,3‐triglucan with β‐configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl‐FH1) complexed with or without oligosaccharides at 1.4–2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl‐FH1 was determined by native single‐wavelength anomalous diffraction. The catalytic domain was composed of two modules, a β‐sandwich fold module, and a right‐handed β‐helix fold module. The structure of the β‐sandwich was similar to those of the carbohydrate‐binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this β‐sandwich fold module is a novel carbohydrate‐binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the β‐helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α‐1,3‐glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site‐directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl‐FH1.Database: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl‐FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo).Enzymes: Agl‐FH1, α‐1,3‐glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11.

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 Å, suggesting the presence of two molecules in an asymmetric unit.

Chitin, a β-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N′-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus β-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2–4, 4-nitrophenyl N-acetyl β-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2–6, but has no detectable activity against 4-nitrophenyl β-d-glucose, 4-nitrophenyl β-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 β-N-acetyl-d-glucosaminidase from Paenibacillus sp.