A biochemical comparison of fungal GH6 cellobiohydrolases

2019 ◽  
Vol 476 (15) ◽  
pp. 2157-2172 ◽  
Author(s):  
Stefan Jarl Christensen ◽  
Kristian Bertel Rømer Mørkeberg Krogh ◽  
Nikolaj Spodsberg ◽  
Kim Borch ◽  
Peter Westh
Keyword(s):  
Adsorption Capacity ◽  
Biochemical Study ◽  
Phylogenetic Analyses ◽  
Glycoside Hydrolase Family ◽  
High Turnover ◽  
Substrate Interactions ◽  
Functional Parameters ◽  
Functional Studies ◽  
Technical Interest ◽  
Biochemical Comparison

Abstract Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

Raw starch degrading α-amylases: an unsolved riddle

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
Author(s):  
Nataša Božić ◽  
Nikola Lončar ◽  
Marinela Šokarda Slavić ◽  
Zoran Vujčić
Keyword(s):  
Glycoside Hydrolase Family ◽  
Food Ingredient ◽  
Raw Starch ◽  
Functional Studies ◽  
Structures And Properties ◽  
Structural Peculiarities ◽  
To Come ◽  
Starch Conversion ◽  
Maltose Syrup ◽  
Hydrolysis Of

AbstractStarch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

scholarly journals Diversification of fungal chitinases and their functional differentiation in Histoplasma capsulatum

2020 ◽  
Author(s):  
Kristie D. Goughenour ◽  
Janice Whalin ◽  
Jason C. Slot ◽  
Chad A. Rappleye
Keyword(s):  
Large Scale ◽  
Histoplasma Capsulatum ◽  
Phylogenetic Analyses ◽  
Domain Architecture ◽  
Functional Differentiation ◽  
Chitinase Activity ◽  
Glycoside Hydrolase Family ◽  
Dimorphic Fungus ◽  
Preferential Expression ◽  
Fungal Chitinases

ABSTRACTChitinases enzymatically hydrolyze chitin, a highly abundant biomolecule with many potential industrial and medical uses in addition to their natural biological roles. Fungi are a rich source of chitinases, however the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase family 18 (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C). The chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade in this larger phylogenetic analysis. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin binding domains (CBD) including the LysM domain do not define specific clades but instead are found more broadly across clades of chitinase enzymes. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade (3 A-V, 1 A-IV, and two A-II), one B clade (B-I), and one formerly classified C clade (C-I) chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. This suggests chitinase activity is variable even within sub-clades and that predictions of functionality require more sophisticated models.

scholarly journals Genetic diversity of CHC22 clathrin impacts its function in glucose metabolism

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Matteo Fumagalli ◽  
Stephane M Camus ◽  
Yoan Diekmann ◽  
Alice Burke ◽  
Marine D Camus ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Phylogenetic Analyses ◽  
Insulin Response ◽  
Membrane Traffic ◽  
Purifying Selection ◽  
Human Populations ◽  
Gene Encoding ◽  
Hunter Gatherers ◽  
Functional Studies ◽  
Glucose Transporter Glut4

CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans. We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene, revealing independent gene loss in at least two vertebrate lineages, after arising from gene duplication. All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin, which mediates endocytosis. For vertebrates retaining CLTCL1, strong evidence for purifying selection supports CHC22 functionality. All human populations maintained two high frequency CLTCL1 allelic variants, encoding either methionine or valine at position 1316. Functional studies indicated that CHC22-V1316, which is more frequent in farming populations than in hunter-gatherers, has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic, altering its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22’s role in metabolism and have potential to differentially influence the human insulin response.

scholarly journals Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungusRasamsonia emersonii

2016 ◽  
Vol 72 (7) ◽  
pp. 860-870 ◽  
Author(s):  
Mikael Gudmundsson ◽  
Henrik Hansson ◽  
Saeid Karkehabadi ◽  
Anna Larsson ◽  
Ingeborg Stals ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Cellulosic Biomass ◽  
Glycoside Hydrolase Family ◽  
Hypocrea Jecorina ◽  
Moderately Thermophilic ◽  
Functional Studies ◽  
Excellent Candidate ◽  
Hydrolase Family

The filamentous fungusHypocrea jecorinaproduces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase fromH. jecorina(HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungusRasamsonia emersonii(ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization ofReCel3A, heterologously produced inH. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies ofReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics ofReCel3A compared withHjCel3A and mixtures containingHjCel3A makeReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

scholarly journals Characterization of abn2 (yxiA), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation

2008 ◽  
Vol 190 (12) ◽  
pp. 4272-4280 ◽  
Author(s):  
José Manuel Inácio ◽  
Isabel de Sá-Nogueira
Keyword(s):  
Bacillus Subtilis ◽  
Mutational Analysis ◽  
Glycoside Hydrolase Family ◽  
Periplasmic Fraction ◽  
Functional Studies ◽  
Glycoside Hydrolase Family 43 ◽  
Transcriptional Start Sites ◽  
Extension Analysis ◽  
Hydrolase Family

ABSTRACT The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-α-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-α-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent Km of 2.0 mg ml−1 and V max of 0.25 mmol min−1 mg−1 at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a σA-dependent and a σH-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

scholarly journals The Chemosensory Transcriptome of a Diving Beetle

2021 ◽  
Vol 9 ◽  
Author(s):  
Nicolas Montagné ◽  
Muriel Jager ◽  
Thomas Chertemps ◽  
Emma Persyn ◽  
Yan Jaszczyszyn ◽  
...  
Keyword(s):  
De Novo ◽  
Human Life ◽  
Phylogenetic Analyses ◽  
Strong Basis ◽  
Male And Female ◽  
Reference Transcriptome ◽  
Functional Studies ◽  
Gene Expansion ◽  
Huge Impact ◽  
Diving Beetle

Insects astoundingly dominate Earth’s land ecosystems and have a huge impact on human life. Almost every aspect of their life relies upon their highly efficient and adaptable chemosensory system. In the air, most chemical signals that are detected at long range are hydrophobic molecules, which insects detect using proteins encoded by multigenic families that emerged following land colonization by insect ancestors, namely the odorant-binding proteins (OBPs) and the odorant receptors (ORs). However, land-to-freshwater transitions occurred in many lineages within the insect tree of life. Whether chemosensory gene repertoires of aquatic insects remained essentially unchanged or underwent more or less drastic modifications to cope with physico-chemical constraints associated with life underwater remains virtually unknown. To address this issue, we sequenced and analyzed the transcriptome of chemosensory organs of the diving beetle Rhantus suturalis (Coleoptera, Dytiscidae). A reference transcriptome was assembled de novo using reads from five RNA-seq libraries (male and female antennae, male and female palps, and wing muscle). It contained 47,570 non-redundant unigenes encoding proteins of more than 50 amino acids. Within this reference transcriptome, we annotated sequences coding 53 OBPs, 48 ORs, 73 gustatory receptors (GRs), and 53 ionotropic receptors (IRs). Phylogenetic analyses notably revealed a large OBP gene expansion (35 paralogs in R. suturalis) as well as a more modest OR gene expansion (9 paralogs in R. suturalis) that may be specific to diving beetles. Interestingly, these duplicated genes tend to be expressed in palps rather than in antennae, suggesting a possible adaptation with respect to the land-to-water transition. This work provides a strong basis for further evolutionary and functional studies that will elucidate how insect chemosensory systems adapted to life underwater.

scholarly journals Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of LIM gene family in Medicago sativa L.

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252213
Author(s):  
Lili Nian ◽  
Xuelu Liu ◽  
Yingbo Yang ◽  
Xiaolin Zhu ◽  
Xianfeng Yi ◽  
...  
Keyword(s):  
Gene Family ◽  
Medicago Sativa ◽  
Phylogenetic Analyses ◽  
Evolutionary Relationship ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Segmental Duplications ◽  
Conserved Motifs ◽  
Functional Studies ◽  
Genome Wide

The LIM (Lin-11, Isl-1 and Mec-3 domains) family is a key transcription factor widely distributed in animals and plants. The LIM proteins in plants are involved in the regulation of a variety of biological processes, including cytoskeletal organization, the development of secondary cell walls, and cell differentiation. It has been identified and analyzed in many species. However, the systematic identification and analysis of the LIM genes family have not yet been reported in alfalfa (Medicago sativa L.). Based on the genome-wide data of alfalfa, a total of 21 LIM genes were identified and named MsLIM01-MsLIM21. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the LIM gene family in alfalfa using bioinformatics methods. The results showed that these MsLIM genes were distributed unequally on 21 of the 32 chromosomes in alfalfa. Gene duplication analysis showed that segmental duplications were the major contributors to the expansion of the alfalfa LIM family. Based on phylogenetic analyses, the LIM gene family of alfalfa can be divided into four subfamilies: αLIM subfamily, βLIM subfamily, γLIM subfamily, and δLIM subfamily, and approximately all the LIM genes within the same subfamily shared similar gene structure. The 21 MsLIM genes of alfalfa contain 10 Motifs, of which Motif1 and Motif3 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of MsLIM genes. Real-time quantitative PCR demonstrated that MsLIM gene expression is induced by low temperature and salt. The present study serves as a basic foundation for future functional studies on the alfalfa LIM family.

scholarly journals Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

2020 ◽  
Author(s):  
Pashupati Bhandari ◽  
Jeffrey P. Tingley ◽  
D. Wade Abbott ◽  
Janet E. Hill
Keyword(s):  
Microbial Community ◽  
Phylogenetic Analyses ◽  
Anaerobic Bacteria ◽  
Glycoside Hydrolase Family ◽  
Vaginal Microbiome ◽  
Multistep Process ◽  
Ph Range ◽  
Culture Supernatants ◽  
Layer Chromatography

AbstractGardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up by bacteria requires the combined activities of several enzymes collectively known as “amylases”. In the present study, glycogen degrading abilities of Gardnerella spp. were assessed. We found that Gardnerella spp. isolates and filtered culture supernatants had amylase activity. Phylogenetic analyses predicted conserved Glycoside Hydrolase family 13 (GH13) members among Gardnerella spp. including a putative α-glucosidase. The gene for this enzyme was cloned and expressed, and recombinant protein was purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4 - 8) with an optimum activity at pH 6-7. Glucose was released from maltose, maltotriose and maltopentose, however, no products were detected on thin layer chromatography (TLC) when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce a secreted α-glucosidase enzyme that can contribute to the complex and multistep process of glycogen breakdown by degrading smaller oligosaccharides into glucose, contributing to the pool of nutrients available to the vaginal microbiota.

scholarly journals Phylogenetic Analysis of the Plant U2 snRNP Auxiliary Factor Large Subunit A Gene Family in Response to Developmental Cues and Environmental Stimuli

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Lu ◽  
Cong Gao ◽  
Yongzhou Wang ◽  
Yingying He ◽  
Junrong Du ◽  
...  
Keyword(s):  
Gene Family ◽  
Plant Species ◽  
Plant Biomass ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
High Temperature Stress ◽  
Metal Exposure ◽  
Functional Studies ◽  
Branch Site ◽  
Recognition Motifs

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3′-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

scholarly journals A synthetic approach for assessing the interplay of form and function in the crocodyliform snout

2019 ◽  
Author(s):  
Stephanie K Drumheller ◽  
Eric W Wilberg
Keyword(s):  
Phylogenetic Analyses ◽  
Hierarchical Cluster ◽  
Synthetic Approach ◽  
Ancestral State ◽  
Crown Group ◽  
Functional Studies ◽  
Form And Function ◽  
And Function ◽  
Specialist Groups ◽  
Prey Items

Abstract Existing classifications of snout shape within Crocodylia are supported by functional studies, but ecological surveys often reveal a higher than expected diversity of prey items within putatively specialist groups, and research into bite force and predation behaviour does not always reveal significant differences between snout shape groups. The addition of more distantly related crocodyliforms complicates the ecomorphological signal, because these groups often occupy a larger area of morphospace than the crown group alone. Here, we present an expanded classification of snout shapes and diets across Crocodyliformes, bringing together geometric morphometrics, non-hierarchical cluster analyses, phylogenetic analyses, ancestral state reconstructions, ecological surveys of diet, and feeding traces from the fossil record to build and test predictive models for linking snout shape and function across the clade. When applied to living members of the group, these new classifications partition out based on differences in predator body mass and maximal prey size. When applied to fossils, these classifications predict potential prey items and identify possible examples of scavenging. In a phylogenetic context, these ecomorphs reveal differences in dietary strategies and diversity within major crocodyliform clades. Taken together, these patterns suggest that crocodyliform diversity, in terms of both morphology and diet, has been underestimated.

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