ArfI and arfII, two genes encoding alpha-L-arabinofuranosidases in Cytophaga xylanolytica.
Ontology highlight
ABSTRACT:
SUBMITTER: Kim KS
PROVIDER: S-EPMC106251 | biostudies-literature | 1998 May
REPOSITORIES: biostudies-literature
Publications
arfI and arfII, two genes encoding alpha-L-arabinofuranosidases in Cytophaga xylanolytica.
Kim K S KS Lilburn T G TG Renner M J MJ Breznak J A JA
Applied and environmental microbiology 19980501 5
arfI encoded the 57.7-kDa subunit of Cytophaga xylanolytica arabinofuranosidase I (ArfI). arfII encoded a 59.2-kDa subunit of ArfII. Products of both cloned genes liberated arabinose from arabinan and arabinoxylan. The deduced amino acid sequences of ArfI and ArfII revealed numerous regions that were identical to each other and to regions of homologous proteins from Bacteroides ovatus, Bacillus subtilis, and Clostridium stercorarium. ...[more]
Similar Datasets
Project description:Species in the extremely thermophilic genus Caldicellulosiruptor can degrade unpretreated plant biomass through the action of multimodular glycoside hydrolases. To date, most focus with these bacteria has been on hydrolysis of glucans and xylans, while the biodegradation mechanism for arabinose-based polysaccharides remains unclear. Here, putative α-l-arabinofuranosidases (AbFs) were identified in Caldicellulosiruptor species by homology to less-thermophilic versions of these enzymes. From this screen, an extracellular XynF was determined to be a key factor in hydrolyzing α-1,2-, α-1,3-, and α-1,5-l-arabinofuranosyl residues of arabinose-based polysaccharides. Combined with a GH11 xylanase (XynA), XynF increased arabinoxylan hydrolysis more than 6-fold compared to the level seen with XynA alone, likely the result of XynF removing arabinofuranosyl side chains to generate linear xylans that were readily degraded. A second AbF, the intracellular AbF51, preferentially cleaved the α-1,5-l-arabinofuranosyl glycoside bonds within sugar beet arabinan. β-Xylosidases, such as GH39 Xyl39B, facilitated the hydrolysis of arabinofuranosyl residues at the nonreducing terminus of the arabinose-branched xylo-oligosaccharides by AbF51. These results demonstrate the separate but complementary contributions of extracellular XynF and cytosolic AbF51 in processing the bioconversion of arabinose-containing oligosaccharides to fermentable monosaccharides.IMPORTANCE Degradation of hemicellulose, due to its complex chemical structure, presents a major challenge during bioconversion of lignocellulosic biomass to biobased fuels and chemicals. Degradation of arabinose-containing polysaccharides, in particular, can be a key bottleneck in this process. Among Caldicellulosiruptor species, the multimodular arabinofuranosidase XynF is present in only selected members of this genus. This enzyme exhibited high hydrolysis activity, broad specificity, and strong synergism with other hemicellulases acting on arabino-polysaccharides. An intracellular arabinofuranosidase, AbF51, occurs in all Caldicellulosiruptor species and, in conjunction with xylosidases, processes the bioconversion of arabinose-branched oligosaccharides to fermentable monosaccharides. Taken together, the data suggest that plant biomass degradation in Caldicellulosiruptor species involves extracellular XynF that acts synergistically with other hemicellulases to digest arabino-polysaccharides that are subsequently transported and degraded further by intracellular AbF51 to produce short-chain arabino sugars.
| S-EPMC5479001 | biostudies-literature
Project description:alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60 degreesC and exhibited stability at pH values from 3 to 7 and at temperatures up to 60 degrees C. The enzymes released arabinose from p-nitrophenyl-alpha-L-arabinofuranoside, O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranose, and arabinose-containing polysaccharides but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L-arabinofuranosyl-(1-->3)-O-b eta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyra nose. alpha-L-Arabinofuranosidase I also released arabinose from O-beta-D-xylopy-ranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl- (1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose. However, alpha-L-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. alpha-L-Arabinofuranosidase I hydrolyzed all linkages that can occur between two alpha-L-arabinofuranosyl residues in the following order: (1-->5) linkage > (1-->3) linkage > (1-->2) linkage. alpha-L-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1-->5) linkage > (1-->2) linkage > (1-->3) linkage. alpha-L-Arabinofuranosidase I preferentially hydrolyzed the (1-->5) linkage of branched arabinotrisaccharide. On the other hand, alpha-L-arabinofuranosidase II preferentially hydrolyzed the (1-->3) linkage in the same substrate. alpha-L-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas alpha-L-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.
| S-EPMC106594 | biostudies-literature
Project description:alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that normally occur between two alpha-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the (1-->3) linkages of the arabinosyl side-chains of methyl 3, 5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the (1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1, 5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.
| S-EPMC1220816 | biostudies-other
Project description:Arabinofuranosidases are important accessory enzymes involved in the degradation of arabinose-containing poly- and oligosaccharides. Two arabinofuranosidases from the recently described novel anaerobic cellulolytic bacterium Acetivibrio mesophilus, designated AmAraf51 and AmAraf43, were heterologously expressed in Escherichia coli and biochemically characterized. AmAraf51 not only removed arabinose moieties at O-3, O-2 and terminal O-5 positions of arabinose-containing oligosaccharides, but also exhibited exo-β-xylosidase side activity. In comparison, AmAraf43 preferably cleaved 1,3-linkages from arabinosyl disubstitutions. AmAraf51 and AmAraf43 demonstrated maximum activity at 70 °C and 57 °C, respectively. Judging from the genetic context and substrate specificity, AmAraf51 may decompose internalized arabino/xylo-oligosaccharides. The embedding of the AmAraf43 gene between genes for several putative xylanolytic enzymes, along with its enzymatic properties suggests that AmAraf43 cleaves arabinose decorations from heteroxylans extracellularly. The enzymes revealed completely converse activity profiles towards arabinan/arabinoxylan: AmAraf51 displayed strong activity on arabinan, while AmAraf43 prefers arabinoxylan. AmAraf51 dramatically stimulated the saccharification level of wheat arabinoxylan (WAX-RS) and sugar beet arabinan when administered along with xylanase M_Xyn10 or arabinanase PpAbn43, respectively. For WAX-RS degradation, the yield of arabinose and xylose was boosted 13.77-fold and 4.96-fold, respectively. The bifunctional activity, thermostability and high catalytic efficiency make AmAraf51 an interesting candidate for industrial applications.
| S-EPMC8307384 | biostudies-literature
Project description:BackgroundThe importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails.ResultsThree fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0-6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s- 1 and 219 s- 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside.ConclusionAll three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
| S-EPMC6472066 | biostudies-literature
Project description:Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.
| S-EPMC11318964 | biostudies-literature
Project description:Putative ?-L-arabinofuranosidases of Oenococcus oeni and Lactobacillus brevis were heterologously expressed and characterized. We report the basic functional properties of the recombinant enzymes in comparison to those of a commercial family 51 arabinosidase of Aspergillus niger.
| S-EPMC3067215 | biostudies-literature
Project description:BackgroundThe biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy.Methodology/principal findingsHere we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently.Conclusions/significanceThe identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events.
| S-EPMC2904702 | biostudies-literature
Project description:Plant α-l-arabinofuranosidases remove terminal arabinose from arabinose-containing substrates such as plant cell wall polysaccharides, including arabinoxylans, arabinogalactans, and arabinans. In plants, de-arabinosylation of cell wall polysaccharides accompanies different physiological processes such as fruit ripening and elongation growth. In this report, we address the diversity of plant α-l-arabinofuranosidases of the glycoside hydrolase (GH) family 51 through their phylogenetic analysis as well as their structural features. The CBM4-like domain at N-terminus was found to exist only in GH51 family proteins and was detected in almost 90% of plant sequences. This domain is similar to bacterial CBM4, but due to substitutions of key amino acid residues, it does not appear to be able to bind carbohydrates. Despite isoenzymes of GH51 being abundant, in particular in cereals, almost half of the GH51 proteins in Poales have a mutation of the acid/base residue in the catalytic site, making them potentially inactive. Open-source data on the transcription and translation of GH51 isoforms in maize were analyzed to discuss possible functions of individual isoenzymes. The results of homology modeling and molecular docking showed that the substrate binding site can accurately accommodate terminal arabinofuranose and that arabinoxylan is a more favorable ligand for all maize GH51 enzymes than arabinan.
| S-EPMC9964162 | biostudies-literature
Project description:The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-β-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and β-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.
| S-EPMC10509167 | biostudies-literature