Modular Hyperthermostable Bacterial Endo-β-1,4-Mannanase: Molecular Shape, Flexibility and Temperature-Dependent Conformational Changes

Abstract

Endo-β-1,4-mannanase from Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of β-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no three-dimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20°C the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan.

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

“…Some hyperthermostable enzymes produced by this microorganism have demonstrated great potential for industrial applications and served as models to investigate structure-function-stability relationships in glycosyl hydrolases Cota et al 2011;Santos et al 2012;Silva et al 2014). For industrial purposes, an enzyme of thermophilic origin can be considered favorable, since elevated temperatures can yield higher substrate solubility, lower viscosity, and thereby lower pumping costs, and limited risks of bacterial contamination (Lundemo et al 2013).…”

Probing the Complex Architecture of Multimodular Carbohydrate-Active Enzymes Using a Combination of Small Angle X-Ray Scattering and X-Ray Crystallography

“…1). In general, high-resolution X-ray crystal structures of the individual modules are fit into the low-resolution SAXS molecular envelopes (~12-20 Å) generating pseudo-atomic resolution models of the ultra multimodular enzymes and their complexes [8][9][10][11]. Determining ultra multimodular protein structures using the combination of XRC and SAXS is an emerging frontier in structural biology research, providing important fundamental insights into full-length CAZymes with industrial, biomedical, and environmental significance.…”

Effect of β‐mannanase domain from Trichoderma reesei on its biochemical characters and synergistic hydrolysis of sugarcane bagasse

“…found that the truncated ThMan5A lacking CBD from Trichoderma harzianum exhibited improved expression efficiency and thermal stability. Furthermore, the linker can optimize the geometry between the other two domains at high temperatures and protect the enzyme , . Therefore, studies relating to enzyme structure can optimize the properties of the enzyme and make it more effective in the catalytic reaction.…”

“…11,12 At present, research on -mannanase has mainly focused on gene cloning, expression, characterization, production and structural analysis. 13,14 Da Silva et al 15 found that endo--1,4-mannanase was composed of a GH5 catalytic domain connected by a linker to a carbohydrate-binding domain (CBD). The function and mechanism of each domain of mannanase was different, with different origins.…”