1,3-Glucanase hydrolyzes -1,3-glucan, an insoluble linear -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 -1,3glucanase 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 Å using synchrotron radiation. The crystals obtained belonged to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2, 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.

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 http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0M (CatAgl‐FH1), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0N (WT/nigerose), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0P (D1045A/nigerose), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0Q (D1068A/nigerose), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0S (D1069A/ nigerose), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0U (D1068A/oligo), and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6K0V (D1069A/oligo). Enzymes Agl‐FH1, α‐1,3‐glucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/59.html) from Paenibacillus glycanilyticus FH11.

S. 4,[5],12:i:-, a monophasic variant of S. enterica serovar Typhimurium, is an important multidrug resistant serovar. Strains of colistin-resistant S. 4,[5],12:i:- have been reported in several countries with patients occasionally had recent histories of travels to Southeast Asia. In the study herein, we investigated the genomes of S. 4,[5],12:i:- carrying mobile colistin resistance (mcr) gene in Thailand. Three isolates of mcr-3.1 carrying S. 4,[5],12:i:- in Thailand were sequenced by both Illumina and Oxford Nanopore platforms and we analyzed the sequences together with the whole genome sequences of other mcr-3 carrying S. 4,[5],12:i:- isolates available in the NCBI Pathogen Detection database. Three hundred sixty-nine core genome SNVs were identified from 27 isolates, compared to the S. Typhimurium LT2 reference genome. A maximum-likelihood phylogenetic tree was constructed and revealed that the samples could be divided into three clades, which correlated with the profiles of fljAB-hin deletions and plasmids. A couple of isolates from Denmark had the genetic profiles similar to Thai isolates, and were from the patients who had traveled to Thailand. Complete genome assembly of the three isolates revealed the insertion of a copy of IS26 at the same site near iroB, suggesting that the insertion was an initial step for the deletions of fljAB-hin regions, the hallmark of the 4,[5],12:i:- serovar. Six types of plasmid replicons were identified with the majority being IncA/C. The coexistence of mcr-3.1 and blaCTX-M-55 was found in both hybrid-assembled IncA/C plasmids but not in IncHI2 plasmid. This study revealed possible transmission links between colistin resistant S. 4,[5],12:i:- isolates found in Thailand and Denmark and confirmed the important role of plasmids in transferring multidrug resistance.

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