Thermophilic Xylanase fromThermomyceslanuginosus:  High-Resolution X-ray Structure and Modeling Studies^,

Abstract

The crystal structure of the thermostable xylanase from Thermomyces lanuginosus was determined by single-crystal X-ray diffraction. The protein crystallizes in space group P21, a = 40.96(4) A, b = 52. 57(5) A, c = 50.47 (5) A, beta = 100.43(5) degrees, Z = 2. Diffraction data were collected at room temperature for a resolution range of 25-1.55 A, and the structure was solved by molecular replacement with the coordinates of xylanase II from Trichoderma reesei as a search model and refined to a crystallographic R-factor of 0.155 for all observed reflections. The enzyme belongs to the family 11 of glycosyl hydrolases [Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788]. pKa calculations were performed to assess the protonation state of residues relevant for catalysis and enzyme stability, and a heptaxylan was fitted into the active-site groove by homology modeling, using the published crystal structure of a complex between the Bacillus circulans xylanase and a xylotetraose. Molecular dynamics indicated the central three sugar rings to be tightly bound, whereas the peripheral ones can assume different orientations and conformations, suggesting that the enzyme might also accept xylan chains which are branched at these positions. The reasons for the thermostability of the T. lanuginosus xylanase were analyzed by comparing its crystal structure with known structures of mesophilic family 11 xylanases. It appears that the thermostability is due to the presence of an extra disulfide bridge, as well as to an increase in the density of charged residues throughout the protein.

Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity

“…Experimentally determined protein structures were obtained from the Protein Data Bank (22), including the following: bovine serum albumin (PDB code 4f5s) (23), the catalytic domains of Acidothermus cellulolyticus endocellulase E1 (Cel5A, PDB code 1c0d) (24), T. reesei cellobiohydrolase I (Cel7A, PDB code 1cel) (25), the family 1 carbohydrate-binding module of T. reesei Cel7A (CBM1, PDB code 1cbh) (26), Thermomyces lanuginosus endo-1,4-␤-xylanase (XynA, PDB code 1yna) (27), T. reesei eno-1,4-␤-xylanase (XynII, PDB code 1enx) (28), and T. reesei acetyl xylan esterase (AxeI, PDB code 1qoz) (29).…”

Thermophilic Fungi: Their Physiology and Enzymes

“…Native xylanase of a strain of H. lanuginosa (T. lanuginosus) (245) and recombinant xylanase of strain DSM 5826 (101) were reported to contain a disulfide bond but no free OSH group. The native xylanase of DSM 5826 contained only a single, catalytically important cysteine but no intramolecular disulfide bond (53).…”

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

“…Structures of covalently attached epoxyalkyl glycosides to the active site residues of XYNII are also available, but these include only one xylose residue (29). Modeling studies with a xyloheptaose in the active site of Thermomyces lanuginosus xylanase (XynA) identify residues involved in subsite Ϫ2 (26). Recently the structure of an inactive mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose has been obtained (17).…”

“…5, structural alignment of the above xylanases with that of A. niger enzyme showed that four of the A. niger xylanase variants (Y10A, Y89A, Y164A, and W172A) have been mutated at positions corresponding to residues that have been involved in discrete subsites of other family 11 xylanases. Tyr 10 corresponds to Trp Xyn11 , all involved in stacking interaction with the xylose ring in substite Ϫ2 of these xylanases (13,16,17,22,26 XYNII , which are thought to determine subsites ϩ1 and ϩ2 of XYNI and XYNII enzyme, respectively (22). Moreover, the oxygen atom OH of residue Tyr 6 is in the same position as atom Glu…”

“…The cleavage by definition takes place between subsites Ϫ1 and ϩ1. Subsites Ϫ3, Ϫ2, and Ϫ1 are well characterized by the inspection of the complex structures (13,16,17), but the characterization of the aglycone subsites is based only on modeling (22,26). Aspergillus niger xylanase is a 20-kDa family 11 xylanase with a pI and a pH optimum of 3.5 (32,33) for which an x-ray crystal structure is available (24).…”