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Updated: Jun 23, 2026

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Published on: May 25, 2018

Understanding how diverse beta-mannanases recognize heterogeneous substrates.

Louise E Tailford¹, Valerie M-A Ducros, James E Flint

¹Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle upon Tyne NE2 4HH, UK.

|May 16, 2009

Summary

This summary is machine-generated.

This study reveals how beta-mannanases (glycoside hydrolase families 5 and 26) distinguish between glucose and mannose in plant glucomannan. Researchers elucidated structural and biochemical mechanisms for substrate specificity in these important polysaccharide-hydrolyzing enzymes.

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Defining Substrate Specificities for Lipase and Phospholipase Candidates

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

Area of Science:

Biochemistry
Structural Biology
Enzymology

Background:

Polysaccharide-hydrolyzing enzymes exhibit variable substrate specificity, particularly with heterogeneous polymers like plant glucomannan.
Beta-mannanases, belonging to glycoside hydrolase (GH) families 5 and 26, are crucial for breaking down glucomannan, a polymer with variable glucose and mannose sequences.

Purpose of the Study:

To investigate the molecular mechanisms underlying substrate specificity in GH5 and GH26 beta-mannanases.
To determine how these enzymes differentiate between glucose and mannose at distal subsites when hydrolyzing glucomannan.

Main Methods:

Biochemical characterization of GH5 (Bacillus agaradhaerens mannanase, BaMan5A) and GH26 (Bacillus subtilis mannanase, BsMan26A) enzymes.
Determination of crystal structures for BaMan5A and BsMan26A.
Site-directed mutagenesis studies on Cellvibrio japonicus GH26 mannanases (CjMan26A).

Main Results:

The GH5 enzyme BaMan5A accommodates both glucose and mannose at its -2 and +1 subsites due to a polar residue interacting with the 2-OH group in either axial or equatorial configuration.
The GH26 enzyme BsMan26A shows strict mannose specificity at negative binding sites, mediated by polar interactions with the axial 2-OH group.
Mutagenesis of CjMan26A demonstrated that specific polar residues are essential for distinguishing mannose from glucose at the -2 subsite.

Conclusions:

GH5 beta-mannanases exhibit broad substrate specificity for variable glucomannan due to flexible substrate binding.
GH26 beta-mannanases achieve tighter mannose recognition through specific polar interactions, highlighting distinct evolutionary strategies for substrate specificity.