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

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Relation http://ir.cftri.com/13951/
https://doi.org/10.1016/j.bbrc.2019.01.085
 
Title How substrate subsites in GH26 endo-mannanase contribute towards
mannan binding.
 
Creator Gaurav Singh, Kaira
Mukesh, Kapoor
 
Subject 05 Enzymes
 
Description 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.
 
Date 2019
 
Type Article
PeerReviewed
 
Format pdf
 
Language en
 
Identifier http://ir.cftri.com/13951/1/BBRC%20Volume%20510%2C%20Issue%203%2C%2012%20March%202019%2C%20Pages%20358-363.pdf
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