Expression and characterization of α-1,3-glucanase from Paenibacillus alginolyticus NBRC15375, which is classified into subgroup 2 (minor group) of GH family 87.

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov., isolated from leaf soil collected in Japan.

Strain CCI5, an oligotrophic bacterium, was isolated from leaf soil collected in Japan. Strain CCI5 grew at temperatures between 25 °C and 43 °C (optimum temperature, 40 °C) and at pHs between 6.0 and 10.0 (optimum pH, 9.0). Its major fatty acids were anteiso-C15:0 and iso-C16:0, and menaquinone 7 was the only detected quinone system. In a phylogenetic analysis based on 16S rRNA gene sequences, strain CCI5 presented as a member of the genus Paenibacillus. Moreover, multilocus sequence analysis based on partial sequences of the atpD, dnaA, gmk, and infB genes showed that strain CCI5 tightly clustered with P. glycanilyticus DS-1T. The draft genome of strain CCI5 consisted of 6,864,972 bp with a G+C content of 50.7% and comprised 6,189 predicted coding sequences. The genome average nucleotide identity value (97.8%) between strain CCI5 and P. glycanilyticus DS-1T was below the cut-off value for prokaryotic subspecies delineation. Based on its phenotypic, chemotaxonomic, and phylogenetic features, strain CCI5 (= HUT-8145T = KCTC 43270T) can be considered as a novel subspecies within the genus Paenibacillus with the proposed name Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov.

Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilyticus FH11.

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 ß-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 ß-sandwich fold module, and a right-handed ß-helix fold module. The structure of the ß-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this ß-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 ß-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.

X-ray crystallographic analysis of the catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis.

α-1,3-Glucanase hydrolyzes α-1,3-glucan, an insoluble linear α-1,3-linked homopolymer of glucose that is found in the extracellular polysaccharides produced by oral streptococci in dental plaque and in fungal cell walls. This enzyme could be of application in dental care and the development of fungal cell-wall lytic enzymes, but its three-dimensional structure has not been available to date. In this study, the recombinant catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus FH11, which is classified into glycoside hydrolase family 87, was prepared using a Brevibacillus choshinensis expression system and purified in a soluble form. Crystals of the purified protein were produced by the sitting-drop vapor-diffusion method. Diffraction data were collected to a resolution of 1.6 Šusing synchrotron radiation. The crystals obtained belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 132.6, c = 76.1 Å. The space group and unit-cell parameters suggest that there is one molecule in the asymmetric unit.

Characterization of α-1,3-glucanase isozyme from Paenibacillus glycanilyticus FH11 in a new subgroup of family 87 α-1,3-glucanase.

Two α-1,3-glucanase isozymes, designated as α-1,3-glucanase 1 (Agl-FH1) and α-1,3-glucanase 2 (Agl-FH2), were purified from the culture medium of Paenibacillus glycanilyticus FH11. Agl-FH1 and Agl-FH2 exhibited similar characteristics such as optimal pH, pH stability, optimal temperature, thermostability, and molecular masses on SDS-PAGE. However, their hydrolysis products of α-1,3-glucan varied somewhat. Agl-FH1 hydrolyzed α-1,3-glucan into a mixture of maltotriose and maltotetraose, and maltotetraose was the major hydrolysis product of Agl-FH2. N-terminal amino acid sequence analysis and LC-MS/MS analysis of trypsin digested fragments revealed several differences between the amino acid sequences of Agl-FH1 and Agl-FH2. Genes of Agl-FH1 and Agl-FH2 were subcloned into an expression plasmid, and both enzymes were successfully expressed in Escherichia coli. The recombinant Agl-FH1 and Agl-FH2 exhibited the same enzymatic properties as those of each wild-type enzyme, and both of the recombinants showed the activity on the protoplast formation of Schizophyllum commune mycelia. A great diversity was detected in the C-terminal region of family 87 α-1,3-glucanases. Compared with Agl-FH2 which is highly sequence-related to the known α-1,3-glucanases, the C-terminal region of Agl-FH1 has only slight similarity to them (approximately 20% identity). Our analysis revealed that Agl-FH1 was the first member of a new subgroup of family 87 α-1,3-glucanases.