Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

Abstract

Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-β-xylanase identified in a microbial consortium

“…The Porod-Debye plot (q 4 x I(q) versus q 4 ) displays a clear plateau, which is consistent with the observation that MetXyn11 is a globularlike protein that lacks disordered regions (Fig. 8C) (Perry and Tainer 2013;De Oliveira et al 2015;Rambo and Tainer 2011). The dimensionless Kratky curve (q 2 x I(q) versus q), which provides a notion about the degree of the particle compactness, has a well-defined maximum very close to 1.1 at qRg = 1.6.…”

“…8D) (Rambo and Tainer 2011;Perry and Tainer, 2013). Furthermore, at qRg> 6 there is a subtle elevated baseline, suggesting that MetXyn11 may exhibit some degree of flexibility (Perry and Tainer 2013;De Oliveira et al 2015). All the experimental SAXS data fitted well to the theoretical SAXS plots generated from the 3D-homology model of MetXyn11 structure.…”

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose

“…On a more subtle level, minor changes in the structure and dynamics of loops in Cel7A cellobiohydrolases have been connected to their catalyticand physical properties (Kurasin and Valjamae, 2011;Momeni et al, 2013Momeni et al, , 2014von Ossowski et al, 2003). These roles of the loops have recurrently led to the suggestion that they make up promising targets for engineering of cellulases and related enzymes for biomass treatment (Adney et al, 2009;Cheng et al, 2012;Chokhawala et al, 2015;Dana et al, 2012;de Oliveira et al, 2015;von Ossowski et al, 2003;Zhang et al, 2000b). However, better understanding of structure-function relationship is needed to enable rational fine tuning of enzyme properties through modifications of the loops.…”

Chemical stability of a cold-active cellulase with high tolerance toward surfactants and chaotropic agent

“…The marked variation in activity observed at pH 4 (Fig. 1A) was probably a consequence of the conformational change that becomes the active site less or more accessible to substrate when compared with the conformation at pH 8 [25], [26].…”