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An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation.

Mamo, Gashaw LU ; Thunnissen, Marjolein LU ; Hatti-Kaul, Rajni LU and Mattiasson, Bo LU (2009) In Biochimie 91(9). p.1187-1196
Abstract
The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2(1)2(1)2(1) and the structure was determined at a resolution of 2.1 A. The enzyme has the common eight-fold... (More)
The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2(1)2(1)2(1) and the structure was determined at a resolution of 2.1 A. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9-9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochimie
volume
91
issue
9
pages
1187 - 1196
publisher
Elsevier
external identifiers
  • wos:000269654100015
  • pmid:19567261
  • scopus:67651236276
ISSN
1638-6183
DOI
10.1016/j.biochi.2009.06.017
language
English
LU publication?
yes
id
fc965025-f2f1-43a6-a90d-910663316bc4 (old id 1453500)
date added to LUP
2009-09-01 09:11:37
date last changed
2017-12-10 03:54:50
@article{fc965025-f2f1-43a6-a90d-910663316bc4,
  abstract     = {The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2(1)2(1)2(1) and the structure was determined at a resolution of 2.1 A. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9-9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH.},
  author       = {Mamo, Gashaw and Thunnissen, Marjolein and Hatti-Kaul, Rajni and Mattiasson, Bo},
  issn         = {1638-6183},
  language     = {eng},
  number       = {9},
  pages        = {1187--1196},
  publisher    = {Elsevier},
  series       = {Biochimie},
  title        = {An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation.},
  url          = {http://dx.doi.org/10.1016/j.biochi.2009.06.017},
  volume       = {91},
  year         = {2009},
}