RESULTS AND DISCUSSION “Refinement” of the primary sequence. Sequence discrepancies were found...

transcript

RESULTS AND DISCUSSION

“Refinement” of the primary sequence.

Sequence discrepancies were found against the available sequence (Bousson &Parriche, 1999, unpublished). Clear electron density maps, supported by multiple sequence alignement, allowed for corrections to be made for residues 245 (Proline) and 217 (Glycine). The terminal glutamine is not seen in the maps. If it is present it is either statically disordered or has considerable thermal motion. See alignement below and figure on the left

Protonation state of the glutamates at the active site

A distance of 5.5Å was observed between OE2 of the acid/base Glu131 and OE2 of the nucleophile Glu237. This is consistent with the retention of the anomeric configuration during xylan hydrolysis. In the active enzyme and in the absence of xylan, the acid/base glutamate should be protonated while Glu237 should be deprotonated (Sinnot, 1990; Davies & Henrissat, 1995). This was shown through a block-diagonal unrestrained refinement (with SHELXH), using one block for all atoms and retaining the positional parameters. The C-O distances and corresponding standard uncertainties confirmed the expected protonation states Mechanism scheme on the left

Side-chain disorder

The clear improvement of the quality of the maps after ADPs were introduced made possible a more precise fitting of side-chain disorder and solvent modelling. Alternate conformations previously not modelled were introduced for 10 residues. Double conformations for residues 223 and 235 modelled by Lo Leggio et al. were removed. The most striking case is tryptophan 275, where alternate conformations were modelled. This residue is thought to be very important in substrate binding to TAXI by closing in on xylan (Lo Leggio et al., in preparation). See structure figures

Quality of the model/Anisotropy

Judging by the overall improvement of the final statistics the anisotropic model seems to fit the data better, as expected considering the mean anisotropy of TAXI. The mean anisotropy is 0.54 for the protein and 0.52 for the solvent, with standard deviations of 0.16 and 0.12, respectively. The distribution of anisotropy among the protein atoms of TAXI was calculated with the program PARVATI. It shows a deviation from the typical more anisotropic distribution curve. This same behaviour has been observed before (Merritt, 1999). It is a fact that the number of structures refined with ADPs is increasing. The growing number of high quality structures will surely bring important information to the parameterisation used in refinement programs and to future studies on anisotropy and refinement protocols. See anisotropic displacement figure and refinement statistics.

INTRODUCTION

Thermoascus aurantiacus Xylanase I has a ()8 TIM-barrel fold and belongs to the family 10 of glycosyl hydrolases (Banner et al., 1975; Jenkins et al., 1995; Pickersgill et al., 1998; Coutinho & Henrissat, 1999). Interest in such enzymes is due to their potential and actual practical applications. Understanding the structure of xylanases and how it correlates to their function is important to support studies aiming at improving and using the properties of these enzymes in practical applications.

TAXI catalyses the hydrolysis of xylan, which represents the major group of hemicelluloses. It is an interesting xylanase, as it has been shown to have a high degree of thermal stability, high activity and high specificity towards xylan. These characteristics may be very useful in future applications in the pulp and paper industries, where reduction in the use of chlorine as a bleaching agent is being imposed by environmental regulations.

DATA USED IN THE REFINEMENT(From 1tax PDB deposition: structure factors were downloaded)

Radiation:(Lo Leggio et all., 1999).

Synchrotron, Beamline 9.6 SRSDaresbury (UK).

Temperature: 293KSpace group: P21

Cell constants: a = 51.04Å , b = 68.30 Å,c = 41.44 Å, = 113.9°

Resolution: 10 – 1.14 ÅNo. Reflections: 80508 (unique)Multiplicity: 2.9I/(I): 9.8 (4.0)Completeness: 85% (77.7%)

In the top left corner a side view of the ()8 TIM-barrel fold of TAXI can be seen. A zoom on the active site shows the side chains of some selected residues (blue) illustrating the environment around the active site glutamates (red). The alternate conformations of tryptophan 275 are coloured in green. The top right corner shows a top view along the barrel.

2Fo-Fc electron density maps contoured at 1 level for the side chains of residues phenylalanine 40 (upper left), lysine 50 (upper right), tyrosine 170 (bottom left) and proline 245 (bottom right). Hydrogens are coloured in white.

The left view shows a zoom on the active site. In the right the same view can be seen but a description of the anisotropic displacement of the side chain atoms of some residues is added: the higher displacements in tryptophan 275 (green) are clearly visible.

Refinement statistics Isotropic AnisotropicAtoms Protein Solvent Hydrogens

2329165

2371 171 2326

Resolution range (Å) 39.5-1.14 10-1.14R-factor ( % for all data) 18 11.1Free R-factor ( % for all data) 20.1 13.7Goodness of Fit (SHELX) 4.63 1.38Restrained Goodness of Fit 4.50 1.18R.m.s. deviations

Bond length (Å) Bond angles (°) Isotropic Beq values Main chain(Å2) Side chain (Å2)

0.0091.493

0.0152.205

1.0443.080

Average B values Main chain (Å2) Side chain (Å2)

B-value11.0813.21

Isot. Beq

12.5117.65

Overall average G-factor

0.36 0.03Double Conformations modelled 8 16

As output by Procheck.

ANISOTROPIC REFINEMENT OF THE STRUCTURE OF ANISOTROPIC REFINEMENT OF THE STRUCTURE OF THERMOASCUS AURANTIACUSTHERMOASCUS AURANTIACUS XYLANASE AT 1.14Å RESOLUTIONXYLANASE AT 1.14Å RESOLUTION

Susana TeixeiraSusana Teixeiraaa, Leila Lo Leggio, Leila Lo Leggiobb, Richard Pickersgill, Richard Pickersgillcc and Christine Cardin and Christine Cardinaa aChemistry Department, University of Reading, Whiteknights, Reading RG6 6AD, England

bCentre for Crystallographic Studies, Chemical Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, DenmarkcMolecular and Cellular Biology, Queen Mary, University of London, Mile End Road, London E1 4NS,England

Chemistry DepartmentFaculty of SciencesUniversity of Reading

Retaining mechanism of the glycosidic bond hydrolysis.

Glu 131

Glu 237

Glu 131

Glu 237

Glu 131

Glu 237

Glu 131

(AcidCatalyst)

(Nucleophile)

-5.5Å

1TAX ------------------------------------------------------------ Q9UQZ4 --------MVRPTILLTSLLLAPFAAAS-------------------------------- 20FOFCH MHTLSVLLALAPVSALAQAPIWGQCGGNGWTGATTCASGLKCEKINDWYYQCVPGSGGSE 60AKXYNA ---------MVQIKAAALAMLFASHVLS-------------------------------- 19MGXYN33A --------MKASSVLLGLAPLAALAAPTP------------------------------- 21 1TAX ------------------ASASAQSVDDQLIKAKARGKGKVYYFGGVATDDQNRLLTTG-KNAAIIQAANNFGFG 41Q9UQZ4 ------------PILEERQAAQSVDDQLIKAKARGKGKVYYFGGVATDDQNRLLTTG-KNAAIIQAADFGFG 67FOFCH PQPSSTQGGGTPQPTGGNSGGTGLDDAKFKAKAKGKGKQYYFGGTEIDDHYHLLNNN-PLINIVKAAQFGFG 119AKXYNA ------------EPIEPRQASVSIDDSKFKAKAHGKGKKYYLGGNIGDDQYTLLTKNSKTPAVIKAADFGFG 67MGXYN33A -----------EAELSARQAQQSIDDALMKAKAKGKGKLYYFGGTATDDQGLLLNTG-KNSAIIKAADFGFG 69

1TAX QVTTPENSMKENSMKWDATEEPSQGNFNFAGADDYLVVNWAAQQNNGKKLIRGHTLIRGHTLVWHSQLPWHSQLPSWVWVTTSIITDDK 101Q9UQZ4 QVTTPENSMKENSMKWDATEEPSQGNFNFAGADDYLVVNWAAQQNNGKKLIRGHTLIRGHTLVWHSQLPWHSQLPSWVWVSSIITDDK 127FOFCH QVTTCENSMKENSMKWDAIEEPSRNSFTFSNADDKVVVDFAATQNNGKKLIRGHTLIRGHTLLWHSQLPWHSQLPQWVWVQNIINDDR 179AKXYNA ALTTPENSMKENSMKWDATEEPSRGQFSFSGSDDYLVVNFAAQSNNNKKLIRGHTLIRGHTLVWHSQLPWHSQLPSWVWVQAIITDDK 127MGXYN33A QVTTPENSMKENSMKCQSLEENTRGQYNWAPADDALVVNFAAVSNNNKKSIRGHTLIRGHTLIWHSQLPWHSQLPGWVWVNNIINDDR 129 1TAX NTLLTNVVMKNHNHITTTLMTRYKGKIKGKIRAWDVVNWDVVN-EEAFFNQNQNGGSLRRSSTVFVFLNVVIGGEDDYIPIAFIAFQTAA 160Q9UQZ4 NTLLTNVVMKNHNHITTTLMTRYKGKIKGKIRAWDVVNWDVVN-EEAFFNEDGGSLRRQTVFVFLNVVIGGEDDYIPIAFIAFQTAA 186FOFCH STLLTAVVIENHNHVKTTMVTRYKGKIKGKILQWDVVNWDVVNNEEIFFAEDGGNLRRDSVFVFSRVVLGGEDDFVGIAFIAFRAAA 239AKXYNA NTLLIEVVMKNHNHITTTVMQHYKGKIKGKIYAWDVVNWDVVN-EEIFFNEDGGSLRRDSVFVFYKVVIGGDDDYVRIAFIAFETAA 186MGXYN33A NQLLTTVVIQNHNHVATTVMGRWKGKIKGKIRAWDVVNWDVVN-EEIFFNEDGGTMRRQSVFVFSRVVLGGEDDFVRIAFIAFEAAA 188

1TAX RRAADPADPNAKLYINDYNLDAKLYINDYNLDSATTYPKKTQA-IVVNRVVKQWWRAAGAGVPIDGIGPIDGIGSQQTHLHLSAGQGG---A 216Q9UQZ4 RRAADPADPNAKLYINDYNLDAKLYINDYNLDSASYPKKTQA-IVVNRVVKQWWRAAGAGVPIDGIGPIDGIGSQQTHLHLSAGQGG---A 242FOFCH RRAADPADPAAKLYINDYNLDAKLYINDYNLDKSDYAKKVTRGMVVAHVVNKWWIAAGAGIPIDGIGPIDGIGSQQGHLHLAAPSGGWNPA 299AKXYNA RRAADPADPNAKLYINDYNLDAKLYINDYNLDSASYPKKLAG-MVVSHVVKKWWIEAGAGIPIDGIGPIDGIGSQQTHLHLSAGGGG---A 242MGXYN33A RRKADPADPNAKLYINDYNLDAKLYINDYNLDRPNAGKKLTKGMVVGHVVKKWWVGAGAGVPIDGIGPIDGIGRQQGHLHLQSGQGG---- 244 1TAX -GGVLNNALPLLLASAGTPEEVAITEELDLDVAGGASPPTDYVNVVNACLACLNVSSSCCVGIGITVWGVWGVADDPDSDSWW 275Q9UQZ4 -SSVLQALPLLLASAGTPEEVAITELDELDVAGGASSSTDYVNVVNACLACLNVQSCCVGIGITVWGVWGVADDPDSWDSW 301FOFCH SGGVPAALRALLAASDAKEEIAITELDELDIAGGASAANDYLTVMNACLACLAVPKCCVGIGITVWGVWGVSDDKDSWDSW 359AKXYNA -GGISGALNALLAGAGTKEEIAVTELDELDIAGGASSSTDYVEVVEACLACLDQPKCCIGIGITVWGVWGVADDPDSWDSW 301MGXYN33A NGGLGQGIKGLLGDSGVKEEVGGNELDELDIQGGNNGGNEFGGGNKACLACLPVPACCVGIGIPAWGVWGVRDDNDSWDSW 304

1TAX RRASTTPLLPLLFDDGNNFNPKPKPAAYNAIVQNNLLQ- 302Q9UQZ4 RRASTTPLLPLLFDDGNNFNPKPKPAAYNAIVQDLLQQQ 329FOFCH RRPGDNPLLPLLYDDSNNYQPKPKAAAFNALANALL-- 385AKXYNA RRSSSTPLLPLLFDDSNNYNPKPKPAAYTAIANALL-- 327MGXYN33A RRPQGNPLLPLLFDDSNNYNPKPKPAAYNSVVQALLK- 331

Red: Residues where mutations had to be made Red: Residues where mutations had to be made Green: Sequence discrepanciesGreen: Sequence discrepancies

Active site

Active site Active site

1TAX Model: Thermoascus aurantiacus xylanase I (PDB acc. code 1TAX; Lo Leggio et al., 1999). Initial sequence: SPTREMBL acc. code Q9UQZ4 (DNA sequencing; Bousson & Parriche, 1999, unpublished). Cel./Xylanase: Fusarium oxysporum cellulase/xylanase (EMBL acc. code FOFCH; Sheppard et al., 1994). Xylanase A: Aspergillus kawachii xylanase A (EMBL acc. code AKXYNA; Ito et al., 1992). Xylanase B: Magnatoporthe grisea xylanase B ( EMBL acc. code MGXYN33A; Wu et al., 1995).

1TAX model

Initial sequence

Cel./Xylanase

Xylanase A

Xylanase B

1TAX modelInitial sequenceCel./XylanaseXylanase AXylanase B

REFERENCES

Banner, D.W., Bloomer, A.C., Petsko, G.A., Phillips, D.C., Pogson, C.I., Wilson, I.A., Corran, P.H., Furth, A.J., Milman, J.D., Offord, R.E., Priddle, J.D., Waley, S.G. (1975). Nature 255, 609-614.

Coutinho, P.M., Henrissat, B. (1999). Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html

Davies, G., Henrissat, B. (1995). Structure 3, 853-859.

Ito, K., Ikemasu, T., Ishikawa, T. (1992). Biosci. Biotechnol. Biochem. 56, 906-912.

Jenkins, J., Lo Leggio, L., Harris, G., Pickersgill, R. (1995). FEBS Letters 362, 281-285.

Lo Leggio, L., Kalogiannis, S., Bhat, M.K., Pickersgill, R.W. (1999). PROTEINS: Structure, Function and Genetics 36, 295-306.

Merritt, E.A., (1999). Acta Cryst. D55, 1109-1117.

Pickersgill, R., Harris, G., Lo Leggio, L., Mayans, O., Jenkins, J. (1998). Biochemical Society Transactions 26, 190-198.

Sheppard, P.O., Grant, F.J., Oort, P.J., Sprecher, C.A., Foster, D.C., Hagen, F.S., Upshall, A., McKnight, G.L., O'Hara, P.J. (1994). Gene 150 (1), 163-167.

Sinnot, M.L. (1990). Chem. Rev. 90, 1171-1202.

Wu, S.C., Kauffmann, S., Darvill, A.G., Albersheim, P. (1995). Mol. Plant Microbe Interact. 8, 506-514.

Teixeira, S., Lo Leggio, L., Pickersgill, R., Cardin, C., (2001). Acta Cryst, in press at the time of the poster printing.

ACKNOWLEDGEMENTS:

ST is grateful to the Chemistry Department of the University of Reading for financial support, and to Dr. E.

Merritt and Dr. G. Sheldrick for their invaluable advice. LL thanks the Danish National Research

Foundation for financial support, Dr. Anne Mølgaard and Henning Osholm Sørensen for helpful

discussions.

RESULTS AND DISCUSSION “Refinement” of the primary sequence. Sequence discrepancies were found...

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