Divalent metal activation of a GH43 β-xylosidase

Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is beta-xylosidase. A gene encoding the 324-amino acid beta-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+ (e.g., 28-fold by Mg2+) and it is inhibited by Cu2+ or Zn2+. Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 beta-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-beta-D-xylopyranoside, and 4-nitrophenyl-alpha-L-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K-i for D-glucose (1.3 M), favorable properties for performance in saccharification reactors. Published by Elsevier Inc.