Inácio, JM, Correia IL, de Sá-Nogueira I.
2008.
Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis. Microbiology. 154:2719-2729., Number 9
AbstractBacillus subtilis produces α-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°C, with p-nitrophenyl-α-L-arabinofuranoside as substrate, gave an apparent Km of 0.498 mM and 0.421 mM, and Vmax of 317 U mg−1 and 311 U mg−1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50°C and 60°C, 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 (1→5) linkages of linear α-1,5-L-arabinan and α-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 (1→2) and (1→3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinose-containing polysaccharides by B. subtilis.
de Sanctis, D, Bento I, Inácio JM, Custódio S, de Sá-Nogueira I, Carrondo MA.
2008.
Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-α-arabinanase from Bacillus subtilis Acta Crystallographica Section F. 64:636–638., Number 7
AbstractTwo Bacillus subtilis extracellular endo-1,5-α-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-α-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 Å, α = 82.3, β = 87.9, ɣ = 63.6°, and P212121, with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 Å. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 Å resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.