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Publisher's version (útgefin grein), The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252TRmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization., Support from the Swedish research council Formas (Grant 2015-769), the BBI, Horizon 2020 project “Macro Cascade” and the Era-nets MBT “Thermofactories” and SusFood2 “ProSeaFood” as well as the NovoNordisk Grant (NNF18OC0034792) is greatly appreciated.

In the Carbohydrate-Active enZymes database (CAZy) glycoside hydrolases (GHs) are classified presently into 156 GH families. In human, there are five known enzymes from the family GH31. Two (MGAM and SI) are intestinal glucosidases involved in saccharide digestion, the acidic glucosidase (GAA) is responsible for glycogen degradation in lysosomes and GANAB (glucosidase II) plays a role in the control of a proper protein folding in the endoplasmic reticulum. The fifth protein is called GANC. It is an α-glucosidase, which is able to release the terminal glucose from maltotriose and glycogen at neutral pH. Its subcellular localization and its physiological function have not been reported in scientific literature yet. Our phylogenetic analysis shows that GANC evolved in early vertebrates from the α-subunit of GANAB. We have thus used an in silico approach to identify changes leading from the α-subunit of GANAB to GANC. We have also searched for residues and regions, which are conserved and under influence of negative selection pressure and which could be important for the function of the enzymes. We have found three residues, which could be responsible for the difference in the substrate specificity reported between the α-subunit of GANAB and GANC. We have also retrieved expression and subcellular localization data, from the Human Protein Atlas database, which shows differences in the expression profiles between GANAB and GANC. Unlike GANAB, GANC seems to be expressed in the nucleoplasm and in the cytoplasm where it colocalizes with actin filaments. The signal sequence and the nuclear localization signal have also been analyzed.

Proteins from the glycoside hydrolase family 3 (GH3) are important bacterial, fungal and plant enzymes involved in cell wall remodeling, energy metabolism and pathogen defense but no animal GH3 proteins have been reported so far. In presented work we use the in silico approach to describe putative GH3 proteins of animals. Based on tertiary structure modeling, domain organization and transcriptomics data analysis, presence of catalytic and substrate binding residues and evolutionary relationship inference, we assume that there is a monophyletic group of GH3 enzymes (probably β-xylosidases) found in various animal taxa with possible role in development.

In an effort to shed more light on the early evolutionary history of the heavy-chain subunits of heteromeric amino acid transporters (hcHATs) rBAT and 4F2hc within the α-amylase family GH13, a bioinformatics study was undertaken. The focus of the study was on a detailed sequence comparison of rBAT and 4F2hc proteins from as wide as possible taxonomic spectrum and enzyme specificities from the α-amylase family. The GH13 enzymes were selected from the so-called GH13 oligo-1,6-glucosidase and neopullulanase subfamilies that represent the α-amylase family enzyme groups most closely related to hcHATs. Within this study, more than 30 hcHAT-like proteins, designated here as hcHAT1 and hcHAT2 groups, were identified in basal Metazoa. Of the GH13 catalytic triad, only the catalytic nucleophile (aspartic acid 199 of the oligo-1,6-glucosidase) could have its counterpart in some 4F2hc proteins, whereas most rBATs contain the correspondences for the entire GH13 catalytic triad. Moreover, the 4F2hc proteins lack not only domain B typical for GH13 enzymes, but also a stretch of ∼ 40 amino acid residues succeeding the β4-strand of the catalytic TIM barrel. rBATs have the entire domain B as well as longer loop 4. The higher sequence–structural similarity between rBATs and GH13 enzymes was reflected in the evolutionary tree. At present it is necessary to consider two different scenarios on how the chordate rBAT and 4F2hc proteins might have evolved. The GH13-like protein from the cnidarian Nematostella vectensis might nowadays represent a protein close to the eventual ancestor of the hcHAT proteins within the GH13 family.