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

In English

Författare

  • Lars Anderson
  • Per Hägglund
  • Dominik Stoll
  • Leila Lo Leggio
  • Torbjörn Drakenberg
  • Henrik Stålbrand

Summary, in English

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.

Publiceringsår

2008

Språk

Engelska

Sidor

86-95

Publikation/Tidskrift/Serie

Biocatalysis and Biotransformation

Volym

26

Issue

1-2

Dokumenttyp

Artikel i tidskrift

Förlag

Taylor & Francis

Ämne

  • Physical Chemistry
  • Biological Sciences

Nyckelord

  • mannose
  • transglycosylation
  • enzyme kinetics
  • beta-mannanase
  • H-1-NMR
  • residue
  • mutarotation

Status

Published

ISBN/ISSN/Övrigt

  • ISSN: 1024-2422