An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation.
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.
Mamo
Gashaw
G
Department of Biotechnology, Center for Chemistry & Chemical Engineering, Lund University, SE-221 00 Lund, Sweden. gashaw.mamo@biotek.lu.se
Thunnissen
Marjolein
M
Hatti-Kaul
Rajni
R
Mattiasson
Bo
B
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D043364:Endo-1,4-beta Xylanases