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Kinetics and stereochemistry of the Cellulomonas fimi beta-mannanase studied using H-1-NMR

Anderson, Lars LU ; Hägglund, Per LU ; Stoll, Dominik; Lo Leggio, Leila; Drakenberg, Torbjörn LU and Stålbrand, Henrik LU (2008) In Biocatalysis and Biotransformation 26(1-2). p.86-95
Abstract
Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was... (More)
Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
mannose, transglycosylation, enzyme kinetics, beta-mannanase, H-1-NMR, residue, mutarotation
in
Biocatalysis and Biotransformation
volume
26
issue
1-2
pages
86 - 95
publisher
Taylor & Francis
external identifiers
  • wos:000253658000012
  • scopus:50549089761
ISSN
1024-2422
DOI
10.1080/10242420701788835
language
English
LU publication?
yes
id
5fb775a3-8357-4e93-85a4-4d680ba84a04 (old id 1193638)
date added to LUP
2008-09-09 09:07:43
date last changed
2017-01-01 04:41:35
@article{5fb775a3-8357-4e93-85a4-4d680ba84a04,
  abstract     = {Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.},
  author       = {Anderson, Lars and Hägglund, Per and Stoll, Dominik and Lo Leggio, Leila and Drakenberg, Torbjörn and Stålbrand, Henrik},
  issn         = {1024-2422},
  keyword      = {mannose,transglycosylation,enzyme kinetics,beta-mannanase,H-1-NMR,residue,mutarotation},
  language     = {eng},
  number       = {1-2},
  pages        = {86--95},
  publisher    = {Taylor & Francis},
  series       = {Biocatalysis and Biotransformation},
  title        = {Kinetics and stereochemistry of the Cellulomonas fimi beta-mannanase studied using H-1-NMR},
  url          = {http://dx.doi.org/10.1080/10242420701788835},
  volume       = {26},
  year         = {2008},
}