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How substrate subsites in GH26 endo-mannanase contribute towards mannan binding.

Gaurav Singh, Kaira and Mukesh, Kapoor (2019) How substrate subsites in GH26 endo-mannanase contribute towards mannan binding. Biochemical and Biophysical Research Communications, 510 (3). pp. 358-363. ISSN 0006-291X

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Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endomannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that �3, �2, �1, þ1 and þ 2 subsites have a major role while, �4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of �1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that �1 and �2 subsite mutants had 27- and 30-fold higher binding affinity (KD) for carob galactomannan when compared with catalytic mutant. The �1 subsite mutant also had highest KD values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher KD) and ivory nut mannan (up to 4.3-fold higher KD) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, �3 mutant displayed 10-fold higher KD for both carob galactomannan and glucomannan while, �4 mutant did not show any noticeable change in KD values when compared to catalytic mutant.

Item Type: Article
Uncontrolled Keywords: GH26 endo-mannanase Isothermal titration calorimetry Substrate subsites Affinity gel electrophoresis Fluorescence titration
Subjects: 600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 12 Mar 2019 11:21
Last Modified: 12 Mar 2019 11:21
URI: http://ir.cftri.com/id/eprint/13951

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