Three‐dimensional structures and functional studies of two GH43 arabinofuranosidases from Weissella sp. strain 142 and Lactobacillus brevis

Abstract

Arabinofuranosidases degrade arabinose-containing oligo and polysaccharides, releasing L-arabinose, which is a potentially useful sugar, shown to reduce glycemic response under certain conditions. Arabinofuranosidases (Arafs) are frequently found in GH43, one of the most common GHfamilies encoded in genomes in gut microbiota, and hence it is of interest to increase understanding of the function of these enzymes in species occurring in the gut. Here we have produced, characterized and solved the three-dimensional structures, at 1.9 and 2.0 A resolution respectively, of two homologous GH43 enzymes, classified under subfamily 26, from Lactobacillus brevis DSM1269 (LbAraf43) and Weissella strain 142 (WAraf43), respectively. The enzymes, with 74% sequence identity to each other, are composed of a single catalytic module with a b-propeller structure typical of GH43, and an active-site pocket with three identifiable subsites (À1, +1, and +2). According to size exclusion chromatography, native WAraf43 is a dimer, while LbAraf43 is a tetramer in solution. Both of them show activity with similar catalytic efficiency on 1,5-a-L-arabinooligosaccharides with a degree of polymerization (DP) of 2-3. Activity is restricted to substrates of low DP, and the reason for this is believed to be an extended loop at the entrance to the active site, creating interactions in the +2 subsite.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera

“…Cellvibrio japonicus [30], Streptomyces avermitilis [32], and Weissella sp. [33]. These ABFs GH43 cannot hydrolyze any branched substrates such as BAOS and sugar beet arabinan.…”

Microwave-assisted xylanase reaction: impact in the production of prebiotic xylooligosaccharides

“…10 BhXyn10A, BhXyn10K80R, and BhHXyn10A enzymes encoded by a synthetic gene (GeneBank accession number: MW311490) were produced in Escherichia coli BL21(DE3) in 2.5 L bioreactors as described in our previous work. 11 Pre-inoculums were prepared in 100 mL of mAT medium 12 with 10 g L À1 glucose as sole carbon source. E. coli strains harboring plasmids pET21::BhXyn10A, and pET21::Bh-Xyn10AK80R were inoculated in culture media supplemented with 100 mg mL À1 ampicillin, while the medium for the strain E. coli 21(DE3) pET28::BhHXyn10A was supplemented with 34 mg mL À1 kanamycin.…”

Taxogenomic assessment and genomic characterisation of Weissella cibaria strain 92 able to metabolise oligosaccharides derived from dietary fibres

“…No growth or production of lactate or SCFAs was detected for the longer AOS with a degree of polymerisation (DP) of 3-5. Utilisation of AOS was expected due to presence of the previously characterised GH43 α-l-arabinofuranosidase from W. cibaria strain 142 (WAraf43) 26 , which has an identical sequence to the corresponding protein in W. cibaria strain 92 (peg 636). WAraf43 in W. cibaria strain 92 was found in a cluster including genes annotated to a transcriptional repressor, a lactose permease and three genes for the conversion of l-arabinose into d-xylulose-5-phosphate, via l-ribulose and l-ribulose-5-phosphate ( Fig.…”

“…3). Interestingly, the WAraf43 enzyme showed activity on both A 2 and A 3 26 while W. cibaria strain 92 did not grow on A 3 ( Supplementary Table S2). This suggests that there is a transportation limitation over the cell membrane for AOS with a DP above 2 as WAraf43 lacks a signal peptide and is thus expected to be intracellular.…”

“…The GH43 α-l-arabinofuranosidase (LVIS_1748) has 75% identity to WAraf43. LbAraf43, a homolog to LVIS_1748, from L. brevis DSM 1269 has been shown to have similar activity to WAraf43, active on AOS with a DP of 2-3 but not on α-1,3-linked l-arabinofuranosides in arabinoxylooligosaccharides (AXOS) 26 . L. brevis ATCC also has a third α-l-arabinofuranosidase, belonging to GH51 (Table 3).…”