Search

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.

The α-amylase is a ubiquitous starch hydrolase catalyzing the cleavage of the α-1,4-glucosidic bonds in an endo-fashion. Various α-amylases originating from different taxonomic sources may differ from each other significantly in their exact substrate preference and product profile. Moreover, it also seems to be clear that at least two different amino acid sequences utilizing two different catalytic machineries have evolved to execute the same α-amylolytic specificity. The two have been classified in the Cabohydrate-Active enZyme database, the CAZy, in the glycoside hydrolase (GH) families GH13 and GH57. While the former and the larger α-amylase family GH13 evidently forms the clan GH-H with the families GH70 and GH77, the latter and the smaller α-amylase family GH57 has only been predicted to maybe define a future clan with the family GH119. Sequences and several tens of enzyme specificities found throughout all three kingdoms in many taxa provide an interesting material for evolutionarily oriented studies that have demonstrated remarkable observations. This review emphasizes just the three of them: (1) a close relatedness between the plant and archaeal α-amylases from the family GH13; (2) a common ancestry in the family GH13 of animal heavy chains of heteromeric amino acid transporter rBAT and 4F2 with the microbial α-glucosidases; and (3) the unique sequence features in the primary structures of amylomaltases from the genus Borrelia from the family GH77. Although the three examples cannot represent an exhaustive list of exceptional topics worth to be interested in, they may demonstrate the importance these enzymes possess in the overall scientific context.

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.

To reveal evolutionary history of maltase gene family in the genus Drosophila, we undertook a bioinformatics study of maltase genes from available genomes of 12 Drosophila species. Molecular evolution of a closely related glycoside hydrolase, the α-amylase, in Drosophila has been extensively studied for a long time. The α-amylases were even used as a model of evolution of multigene families. On the other hand, maltase, i.e., the α-glucosidase, got only scarce attention. In this study, we, therefore, investigated spatial organization of the maltase genes in Drosophila genomes, compared the amino acid sequences of the encoded enzymes and analyzed the intron/exon composition of orthologous genes. We found that the Drosophila maltases are more numerous than previously thought (ten instead of three genes) and are localized in two clusters on two chromosomes (2L and 2R). To elucidate the approximate time line of evolution of the clusters, we estimated the order and dated duplication of all the 10 genes. Both clusters are the result of ancient series of subsequent duplication events, which took place from 352 to 61 million years ago, i.e., well before speciation to extant Drosophila species. Also observed was a remarkable intron/exon composition diversity of particular maltase genes of these clusters, probably a result of independent intron loss after duplication of intron-rich gene ancestor, which emerged well before speciation in a common ancestor of all extant Drosophila species.

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.

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.