Salt bridges are pivotal for the kinetic stability of GH26 endomannanase (ManB-1601).

Gaurav Singh, Kaira and Mukesh, Kapoor and Usharani, D. (2019) Salt bridges are pivotal for the kinetic stability of GH26 endomannanase (ManB-1601). International Journal of Biological Macromolecules, 133. pp. 1236-1241. ISSN 0141-8130

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Abstract

Hitherto, how salt bridges contribute towards the structure-function of endo-mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo-mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endomannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and E171/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (ΔTm 0 to -50C) and conformational stability (ΔΔG -0.37 to -1.25 Kcal mol-1) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T1/2) at 660C (1.1 to 6-fold) and 700C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural elements for enzyme functioning by contributing towards kinetic stability.

Item Type:	Article
Uncontrolled Keywords:	Conformational stability; GH26 endo-mannanase; Kinetic stability; Molecular dynamics; Salt bridges; Thermal stability
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology 600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions:	Food Safety Analytical Quality Control Lab Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	03 Jun 2019 04:35
Last Modified:	03 Jun 2019 04:35
URI:	http://ir.cftri.res.in/id/eprint/14109

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