Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

Abstract

Bacillus subtilis produces a-L-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (.250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 6C, with p-nitrophenyl-a-L-arabinofuranoside as substrate, gave an apparent K m of 0.498 mM and 0.421 mM, and V max of 317 U mg "1 and 311 U mg "1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 6C and 60 6C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1A5) linkages of linear a-1,5-L-arabinan and a-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1A2) and (1A3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinosecontaining polysaccharides by B. subtilis.

A Multitask ATPase Serving Different ABC-Type Sugar Importers in Bacillus subtilis

“…The three proteins, which are encoded by the arabinose metabolic operon araABDLMNPQ-abfA, previously were shown to be dispensable for arabinose utilization in a strain bearing a large deletion comprising all genes downstream from araD (23). However, growth experiments indicated a possible involvement in the utilization of arabinose oligomers (10). To confirm this hypothesis, an in-frame deletion mutation in the araN gene was generated by allelic replacement to minimize the polar effect on the genes of the araABDLMNPQ-abfA operon, which is located downstream of araN (Fig.…”

“…AraN is the BPD, and AraP and AraQ are the TMDs. This transporter, which lacks the NBD protein partner, was proposed to be involved in the uptake of arabinose oligomers mainly by genomic context and in silico analysis (10,11,23).…”

The l -Arabinan Utilization System of Geobacillus stearothermophilus

“…The resulting products, arabinose and arabinose oligomers, are transported by specific transport systems, AraE, a proton symporter, and AraNPQ, an ABCtype transporter for arabino-oligosaccharides, and presumably an additional, unidentified transporter (22). Inside the cell, arabino oligomers are further hydrolyzed by the concerted action of the two ␣-L-arabinofuranosidases (35).…”

“…Lastly, aerobic bacteria, such as Bacillus and Cellvibrio spp., secrete only a limited number of endo-type enzymes that degrade the polysaccharide backbone into relatively large oligosaccharides. The final breakdown of these oligosaccharides is carried out by cell-associated or intracellular enzymes (35,50,67). The latter strategy, which is presented elegantly in the Geobacillus spp.…”

The abp gene in Geobacillus stearothermophilus T‐6 encodes a GH27 β‐l‐arabinopyranosidase

“…These enzymes are two a-L-arabinofuranosidases (AbfA and AbfB) and one arabinanase (AbnB). AbfA and AbfB remove a-1,2-and a-1,3-linked arabinofuranoside substitutions [13][14][15][16][17]. The role of the araJKLMN genes in the arabinan system is not fully understood and they are likely to constitute a novel utilization pathway for pentoses in G. stearothermophilus [5,18].…”