Structural and functional insights into extreme thermal stability and activity of two GH12 domains of a multidomain glycosidase from a hyperthermophilic euryarchaeon.

Bacteria and fungi are well known for efficient degradation of plant polysaccharides thanks to various enzymes involved in plant cell wall decomposition. However, little is known about the role of archaea in this process or the repertoire and features of their polysaccharide-degrading enzymes. In our previous work, we discovered an archaeal multidomain glycosidase (MDG) composed of three catalytic domains (GH5 and two GH12) and two cellulose-binding modules (CBM2). The recombinant MDG and individual GH5 catalytic domain were active against cellulose and a number of other polysaccharides at a wide range of temperatures, with optimum temperatures (T(opt)) of 60 °C and 80 °C, respectively. The present study was focused on the characterization of two GH12 domains of the MDG. Purified recombinant TMDG_GH12-1 and TMDG_GH12-2 proteins were active as individual enzymes but exhibited distinct catalytic properties. Both enzymes were thermostable and active at extremely high temperatures: TMDG_GH12-1 was active at 40-130 °C (T(opt) 100 °C), and its half-life (t(½)) at 100 °C was 42 h, which makes it one of the most thermostable glycosidases known so far, whereas TMDG_GH12-2 was active at 50-100 °C (T(opt) 90 °C) with t(½) at 100 °C being 30 min. Phylogenetic and structural analysis of both TMDG_GH12 proteins together with molecular docking and site-directed mutagenesis suggested that the presence of two disulfide bridges and the W → Q mutation in the active site contribute to the exceptional thermostability of TMDG_GH12-1. Further structural and mutational studies of the TMDG_GH12-1 domain will help to gain a better understanding of the molecular mechanisms of its extraordinary thermostability and substrate specificity.