The Bifidobacterium adolescentis BAD_1527 gene encodes GH43_22 α-L-arabinofuranosidase of AXH-m type.

Bifidobacterium adolescentis gene BAD_1527 has previously been suggested to code for a ß-xylosidase (Kobayashi et al., Mar Drugs 18:174, 2020). Our detailed investigation of the substrate specificity of the GH43_22 protein using a wide spectrum of natural and artificial substrates showed that the enzyme hydrolyzed neither linear xylooligosaccharides nor glucuronoxylan. Xylose was released only from the artificial 4-nitrophenyl ß-D-xylopyranoside (1.58 mU/mg). The corresponding α-L-arabinofuranoside was by three orders of magnitude better substrate (2.17 U/mg). Arabinose was the only monosaccharide liberated from arabinoxylan and α-1,3- or α-1,2-singly arabinosylated xylooligosaccharides. Moreover, the enzyme efficiently debranched sugar beet arabinan and singly arabinosylated α-1,5-L-arabinooligosaccharides, although short linear α-1,5-L-arabinooligosaccharides were also slowly degraded. On the other hand, debranched arabinan, arabinogalactan as well as 2,3-doubly arabinosylated main chain residues of arabinan and arabinoxylan did not serve as substrates. Thus, the enzyme encoded by the BAD_1527 gene is a typical α-L-arabinofuranosidase of AXH-m specificity.

Sugarcane bagasse derived xylooligosaccharides produced by an arabinofuranosidase/xylobiohydrolase from Bifidobacterium longum in synergism with xylanases.

Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

Optimization of α-L-arabinofuranosidase CcABF on clarification and beneficial active substances in fermented ginkgo kernel juice by artificial neural network and genetic algorithm.

This study aimed at using α-L-arabinofuranosidase CcABF to improve the clarity and active substances in fermented ginkgo kernel juice by artificial neural network (ANN) modeling and genetic algorithm (GA) optimization. A credible three-layer feedforward ANN model was established to predict the optimal parameters for CcABF clarification. The experiments proved the highest transmittance of 89.40% for fermented ginkgo kernel juice with this understanding, which exhibited a 25.56% increase over the unclarified group. With the clarification of CcABF, the antioxidant capacity in juice was enhanced with the increase of total phenolic and flavone contents, and the maximum DPPH and hydroxyl radical scavenging rates were increased by 89.71% and 26.65%, respectively. The contents of toxic ginkgolic acids declined markedly, while the active ingredients of ginkgetin and ginkgolide B showed a modest increase. Moreover, changes in free amino acids and volatile compounds improved the nutritive value and flavor of clarified fermented ginkgo kernel juice.

Molecular cloning, expression, purification, and characterization of Bacillus subtilis hydrolyzed ginsenoside Rc of α-L-arabinofuranosidase in Escherichia coli.

The α-L-arabinofuranosidase enzyme plays a crucial role in the degradation of ginsenosides. In this study, we successfully cloned and expressed a novel α-L-arabinofuranosidase bsafs gene (1503 bp, 501 amino acids, 55 kDa, and pI = 5.4) belonging to glycosyl hydrolase (GH) family 51 from Bacillus subtilis genome in Escherichia coli BL21 cells. The recombinant protein Bsafs was purified using Ni2+ sepharose fastflow affinity chromatography and exhibited a specific activity of 2.91 U/mg. Bsafs effectively hydrolyzed the α-L-arabinofuranoside at C20 site of ginsenoside Rc to produce Rd as the product. The Km values for hydrolysis of pNP-α-L-arabinofuranoside (pNPαAraf) and ginsenoside Rc were determined as 0.74 and 4.59 mmol/L, respectively; while the Vmax values for these substrates were found to be 24 and 164 µmol/min/mg, respectively; furthermore, the Kcat values for these enzymes were calculated as 22.3 and 1.58 S-1 correspondingly.

Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Microbial α-L-arabinofuranosidases: diversity, properties, and biotechnological applications.

Arabinoxylans (AXs) are hemicellulosic polysaccharides consisting of a linear backbone of ß-1,4-linked xylose residues branched by high content of α-L-arabinofuranosyl (Araf) residues along with other side-chain substituents, and are abundantly found in various agricultural crops especially cereals. The efficient bioconversion of AXs into monosaccharides, oligosaccharides and/or other chemicals depends on the synergism of main-chain enzymes and de-branching enzymes. Exo-α-L-arabinofuranosidases (ABFs) catalyze the hydrolysis of terminal non-reducing α-1,2-, α-1,3- or α-1,5- linked α-L-Araf residues from arabinose-substituted polysaccharides or oligosaccharides. ABFs are critically de-branching enzymes in bioconversion of agricultural biomass, and have received special attention due to their application potentials in biotechnological industries. In recent years, the researches on microbial ABFs have developed quickly in the aspects of the gene mining, properties of novel members, catalytic mechanisms, methodologies, and application technologies. In this review, we systematically summarize the latest advances in microbial ABFs, and discuss the future perspectives of the enzyme research.

Characterization of a novel thermostable α-l-arabinofuranosidase for improved synergistic effect with xylanase on lignocellulosic biomass hydrolysis without prior pretreatment.

Utilizing thermostable enzymes in biomass conversion processes presents a promising approach to bypass pretreatment, garnering significant attention from the biorefinery industry. A novel discovered α-l-arabinofuranosidase, Abf4980, exhibits exceptional thermostability by maintaining full activity after 24 h of incubation at 70 °C. It effectively acts on polyarabinosides, cleaving α-1,2- and α-1,3-linked arabinofuranose side chains from water-soluble wheat arabinoxylan while releasing xylose. When synergistically combined with the thermostable bifunctional xylanase/ß-glucanase CbXyn10C from Caldicellulosiruptor bescii at an enzyme-activity ratio of 6:1, Abf4980 achieves the highest degradation efficiency for wheat arabinoxylan. Furthermore, Abf4980 and CbXyn10C demonstrated remarkable efficacy in hydrolyzing unmodified wheat bran and corn cob to generate arabinose and xylooligosaccharides. This discovery holds promising opportunities for improving the efficiency of lignocellulosic biomass conversion into fermentable sugars.

Highly efficient synergistic activity of an α-L-arabinofuranosidase for degradation of arabinoxylan in barley/wheat.

Here, an α-L-arabinofuranosidase (termed TtAbf62) from Thermothelomyces thermophilus is described, which efficiently removes arabinofuranosyl side chains and facilitates arabinoxylan digestion. The specific activity of TtAbf62 (179.07 U/mg) toward wheat arabinoxylan was the highest among all characterized glycoside hydrolase family 62 enzymes. TtAbf62 in combination with endoxylanase and ß-xylosidase strongly promoted hydrolysis of barley and wheat. The release of reducing sugars was significantly higher for the three-enzyme combination relative to the sum of single-enzyme treatments: 85.71% for barley hydrolysis and 33.33% for wheat hydrolysis. HPLC analysis showed that TtAbf62 acted selectively on monosubstituted (C-2 or C-3) xylopyranosyl residues rather than double-substituted residues. Site-directed mutagenesis and interactional analyses of enzyme-substrate binding structures revealed the catalytic sites of TtAbf62 formed different polysaccharide-catalytic binding modes with arabinoxylo-oligosaccharides. Our findings demonstrate a "multienzyme cocktail" formed by TtAbf62 with other hydrolases strongly improves the efficiency of hemicellulose conversion and increases biomass hydrolysis through synergistic interaction.

Biochemical characterization of bifunctional enzymatic activity of a recombinant protein (Bp0469) from Blautia producta ATCC 27340 and its role in the utilization of arabinogalactan oligosaccharides.

Human consumption of larch arabinogalactan has a significant effect on enhancing probiotic microflora in the gut, and it also promotes the production of short-chain fatty acids. Bacterial members of Lachnospiraceae family are important and play significant roles in maintaining our gut health. However, it is less known about biochemistry of members of this family by which they utilize non-cellulosic fiber in the gut. For enhancing this understanding, we studied that B. producta ATCC 27340 grew on arabinogalactan oligosaccharides (AGOs) as compared to polysaccharide form of arabinogalactan. Recombinant protein (Bp0469) was heterologously expressed in Escherichia coli BL21 (DE3) and revealed the optimum pH and temperature at 7.4 in phosphate buffer and 45 °C, respectively. Catalytic efficiency of recombinant Bp0469 for p-nitrophenyl (pNP)-α-L-arabinofuranoside was about half of pNP-ß-D-galactopyranoside. It also cleaved natural substrates (lactose, arabinobiose and 3-O-(ß-d-galactopyranosyl)-d-galactopyranose) and characterized AGOs in this study. Based on genomic, structural models, and biochemical characteristics, identified Bp0469 is a peculiar enzyme with two distinct domains that cleave α1-5 linked arabinobiose and ß-D-Galp-1-3/4 linkages. Overall, the study enhances the knowledge on nutritional perspective of B. producta ATCC 27340 for thriving on non-cellulosic biomass, and identified enzyme can also be used for producing industrial important AGOs.

Expression and characterization of α-L-arabinofuranosidase derived from Aspergillus awamori and its enzymatic degradation of corn byproducts with xylanase.

In this study, α-L-arabinofuranosidase (AF) from Aspergillus awamori was heterologously expressed in Pichia pastoris X33, with a 1-fold increase in AF activity after codon and vector optimization. AF remained stable at 60-65 °C and displayed a broad pH stability range of 2.5-8.0. It also demonstrated considerable resistance to pepsin and trypsin. Furthermore, compared with xylanase alone, AF with xylanase exhibited a marked synergistic effect in the degradation of expanded corn bran, corn bran, and corn distillers' dried grains with solubles, reducing sugars by 3.6-fold, 1.4-fold, and 6.5-fold, respectively, with the degree of synergy increasing to 4.61, 2.44, and 5.4, respectively, while in vitro dry matter digestibility values were 17.6%, 5.2%, and 8.8%, respectively. After enzymatic saccharification, corn byproducts were converted to prebiotic xylo-oligosaccharides and arabinoses, thereby demonstrating the favorable properties of AF in the degradation of corn biomass and its byproducts.

Recombinant Production and Biochemical Characterization of Thermostable Arabinofuranosidase from Acidothermophilic Alicyclobacillus Acidocaldarius.

The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius.

Characterization of an α-L-Arabinofuranosidase GH51 from the Brown-rot Fungus Gloeophyllum trabeum.

Woody biomass is anticipated to be a resource for a decarbonized society, but the difficulty of isolating woody components is a significant challenge. Brown-rot fungi, a type of wood rotting fungi, decompose hemicellulose particularly efficiently. However, there are few reports on the hemicellulases from brown-rot fungi. An α-L-arabinofuranosidase belonging to glycoside hydrolase family 51 (GH51) from the brown-rot fungus Gloeophyllum trabeum (GtAbf51A) was cloned and characterized in the present study. Analyses of the phylogeny of GH51 enzymes in wood rotting fungi revealed the existence of two groups, intercellular and extracellular enzymes. After deglycosylation, the recombinant GtAbf51A produced by Pichia pastoris appeared on SDS-PAGE as approximately 71,777 daltons, which is the expected molecular weight based on the amino acid sequence of GtAbf51A. Maximum enzyme activity occurred between pH 2.2 and 4.0 and at 50 °C, while it was stable between pH 2.2 and 10.0 and up to 40 °C. Due to the presence of a signal peptide, GtAbf51A was thought to hydrolyze polysaccharide containing arabinose. However, the hydrolysis rate of arabinosyl linkages in polysaccharides was only 3-5 % for arabinoxylan and 18 % for arabinan. GtAbf51A, in contrast, efficiently hydrolyzed arabinoxylooligosaccharides, particularly O-α-L-arabinofuranosyl-(1→3)-O-ß-D-xylopyranosyl-(1→4)-ß-D-xylopyranose, which is the principal product of GH10 ß-xylanase. These data suggest that GtAbf51A cooperates with other xylan-degrading enzymes, such as ß-xylanase, to degrade xylan in nature.

The In Silico Characterization of Monocotyledonous α-l-Arabinofuranosidases on the Example of Maize.

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.

Salmonella-liberated dietary L-arabinose promotes expansion in superspreaders.

The molecular understanding of host-pathogen interactions in the gastrointestinal (GI) tract of superspreader hosts is incomplete. In a mouse model of chronic, asymptomatic Salmonella enterica serovar Typhimurium (S. Tm) infection, we performed untargeted metabolomics on the feces of mice and found that superspreader hosts possess distinct metabolic signatures compared with non-superspreaders, including differential levels of L-arabinose. RNA-seq on S. Tm from superspreader fecal samples showed increased expression of the L-arabinose catabolism pathway in vivo. By combining bacterial genetics and diet manipulation, we demonstrate that diet-derived L-arabinose provides S. Tm a competitive advantage in the GI tract, and expansion of S. Tm in the GI tract requires an alpha-N-arabinofuranosidase that liberates L-arabinose from dietary polysaccharides. Ultimately, our work shows that pathogen-liberated L-arabinose from the diet provides a competitive advantage to S. Tm in vivo. These findings propose L-arabinose as a critical driver of S. Tm expansion in the GI tracts of superspreader hosts.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases.

BACKGROUND: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions. RESULTS: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose-xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose-xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO-xylan interaction, because debranching changes the architecture of the xylan surface. CONCLUSIONS: Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Structural and functional analyses of GH51 alpha-L-arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability.

Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid-base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C-C bond between C171 and C213 by becoming closer to each other.

Bifidobacterial GH146 ß-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-ß-l-Araf-(1→2)-ß-l-Araf-(1→2)-ß-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 ß-l-arabinobiosidase (HypBA2), producing ß-l-Araf-(1→2)-l-Ara (ß-l-arabinobiose) and mono-ß-l-Araf linked to the HRGP backbone. In bifidobacteria, the ß-l-arabinobiose is then hydrolyzed by GH127 ß-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 ß-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Mechanism-based inhibition of GH127/146 cysteine glycosidases by stereospecifically functionalized l-arabinofuranosides.

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-l-arabinofuranosidase (Arafase) - HypBA1 - has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as l-Araf-haloacetamides have been carried out. The α-l-Araf-azide derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.