High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-l-arabinofuranosidase activity

摘要

A gene from the heterotrophic, halothermophilic marine bacterium has been cloned and overexpressed in . This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by . The crystal structure of the glycosidase was determined by molecular replacement and refined at 1.10 Å resolution. As for other GH43 members, the enzyme folds as a five-bladed -propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 NaCl and Mn at pH 6.5. The gene encoding the GH43 enzyme has previously been annotated as a putative --arabinofuranosidase. Activity was detected with -nitrophenyl---arabinofuranoside as a substrate, and therefore the name Araf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 NaCl and Mn at pH 6.5, giving a specific activity of 20-36 µmol min mg. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of Araf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.