Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-β-d-mannanase

Abstract

The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

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

“…Furthermore, for industrial applications, β-mannanases stable and functional at high temperatures offer substantial techno-economical advantages. A recent study reported that the deletion of the linker plus TpManCBM27 from TpMan has no statistically relevant effect on the catalytic efficiency upon both mannan and glucomannan substrates [10]. For both substrates, the truncated catalytic domain showed higher V max .…”

“…Thermotoga petrophila strain RKU-1 is a hyperthermophilic bacterium isolated from the production fluid of the Kubiki oil reservoir in Niigata (Japan), which grows optimally at pH 7 and 80°C [9]. This bacterium produces a repertoire of hyperthermostable enzymes of great industrial interest, including cellulases, arabinofuranosidases, arabinases and mananases, and it has proved to be a suitable source of enzymes for biotechnological applications and protein engineering of glycoside hydrolases [10–13]. …”

“…The endo-β-1, 4-mannanases (EC 3.2.1.78) are enzymes that catalyze the hydrolysis of β-1, 4-mannoside linkages in a variety of mannose-containing polysaccharides, including glucomannans and galactomannans [7,10]. These enzymes have been widely studied owing to their roles in seed germination and fruit ripening [7].…”

“…petrophila (TpMan) is an enzyme composed of a GH5 catalytic domain (TpManGH5) and a carbohydrate-binding domain (TpManCBM27) connected through a linker [10,13]. The structure of TpManGH5 is a canonical (α/β) 8 -barrel scaffold surrounded by loops and short helices that form the catalytic interface [10]. In a recent study, we presented the low-resolution model of the intact TpMan studied by small angle X-ray scattering [13].…”

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).…”