Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate
10.1126/science.280.5363.578
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl–transfer RNA synthetase, activates not only the cognate substrate l -isoleucine but also the minimally distinct l -valine in the first, aminoacylation step. Then, in a second, “editing” step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a “double-sieve” mechanism has already been proposed. The present crystal structures of the synthetase in complexes with l -isoleucine and l -valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular β-barrel domain that protrudes from the aminoacylation domain.

Bibliography

Nureki, O., Vassylyev, D. G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T. L., Schimmel, P., & Yokoyama, S. (1998). Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate. Science, 280(5363), 578–582.

Authors 10
  1. Osamu Nureki (first)
  2. Dmitry G. Vassylyev (additional)
  3. Masaru Tateno (additional)
  4. Atsushi Shimada (additional)
  5. Takashi Nakama (additional)
  6. Shuya Fukai (additional)
  7. Mitiko Konno (additional)
  8. Tamara L. Hendrickson (additional)
  9. Paul Schimmel (additional)
  10. Shigeyuki Yokoyama (additional)
References 32 Referenced 278
  1. 10.1016/S0021-9258(18)96841-9
  2. A. Fersht Enzyme Structure and Mechanism (Freeman New York 1985); Biochemistry 16 1025 (1977); A. R. Fersht and C. Dingwall ibid. 18 2627 (1979). (10.1021/bi00579a030)
  3. Freist W., Pardowitz I., Cramer F., Biochemistry24, 7014 (1985). (10.1021/bi00345a040) / Biochemistry by Freist W. (1985)
  4. Freist W., ibid28, 6787 (1989). / ibid by Freist W. (1989)
  5. L. Pauling in Festschrift Arthur Stroll (Birkhäuser-Verlag Basel Switzerland 1957) pp. 597–602.
  6. 10.1126/science.8146659
  7. 10.1016/S0021-9258(19)45304-0
  8. 10.1021/bi00035a028
  9. 10.1038/384033b0
  10. The T. thermophilus IleRS gene was cloned into a T7 polymerase expression vector and was overexpressed in E. coli strain JM109(DE3) (S. Fukai et al. in preparation). The recombinant protein was purified by a combination of heat treatment anion-exchange chromatography and reversed-phase fast performance liquid chromatography. Crystals were grown at 20°C by the hanging-drop vapor diffusion method (protein concentration of 20 mg/ml) against a reservoir solution containing 12% polyethylene glycol 4000 6% isopropanol 1% 2-methyl-pentane-2 4-diol and 60 mM Hepes (pH 7.5). Two mutant proteins with substitutions of Cys for Ala or Lys were expressed purified and used for the preparation of mercury derivatives. Furthermore we incorporated selenium into the IleRS by preparing the IleRS from the E. coli Met-auxotroph B834 strain cultured in the presence of selenomethionine. Crystals of the Cys- and selenomethionine-substituted proteins were grown by cross seeding. Heavy atom derivatives were prepared by cocrystallization of the Cys mutants and methylmercurichloride. Native and heavy atom derivative data were collected to 2.5 Å with a Weissenberg camera for macromolecules installed on the beam line 6A2 at the Photon Factory (Tsukuba Japan). The data set for crystal of selenomethionine-substituted IleRS was collected in-house on a Raxis-IIc (Rigaku Tokyo Japan) image plate detector mounted on a Rigaku x-ray source. All of the data were processed with the DENZO and SCALEPACK programs (25). Subsequent calculations were carried out with the CCP4 program suite (26). Initial mercury sites in the derivative of the K3C mutant were determined with the RSPS program (26) from an isomorphous difference Patterson map. The phases from the mercury derivative were used to locate the positions of other mercury and selenium atoms by difference Fourier analysis. Heavy-atom parameters were refined with the MLPHARE (26) program. The overall figure of merit from 50 to 3.0 Å resolution is 0.74. After density modification with solvent flattening and histogram matching in the program DM (26) and solvent flipping in the SOLOMON (27) program the atomic model of residues 1 to 818 could be built with the program O (28). Crystallographic positional and slow-cooling refinement was carried out with X-PLOR (29). The Ramachandran plot analysis with the PROCHECK (30) program showed that 98.2% of the residues in the present structure are in the most favorable and the additionally allowed regions. We obtained the Ile and Val complexes by transferring the crystals to a harvest buffer containing 10 mM amino acid substrates. The data sets were collected on a Raxis-IIc image plate detector. After rigid-body refinement in the program AMORE (26) the R factors were reduced.
  11. 10.1038/347203a0
  12. S. Fukai et al. unpublished data.
  13. O. Nureki et al. data not shown.
  14. 10.1126/science.3306924
  15. O. Nureki et al. unpublished data.
  16. Rawlings N. D., Barrett A. J., Methods Enzymol.244, 19 (1994). (10.1016/0076-6879(94)44004-2) / Methods Enzymol. by Rawlings N. D. (1994)
  17. Strynadka N. C., et al., Nature359, 700 (1992). (10.1038/359700a0) / Nature by Strynadka N. C. (1992)
  18. Miller M., Rao M., Wlodawer A., Gribskov M. R., FEBS Lett.328, 275 (1993). (10.1016/0014-5793(93)80943-O) / FEBS Lett. by Miller M. (1993)
  19. Charging assays: Mutants were examined for their ability to catalyze the formation of Ile-tRNA Ile under standard charging assay conditions. Final concentrations in each reaction mixture were 1 nM enzyme 1.8 μM tRNA Ile 20 mM Hepes (pH 7.5) 100 μM EDTA 150 mM NH 4 Cl bovine serum albumin (10 μg/ml) 2 mM ATP 4 mM MgCl 2 and 20 μM l -isoleucine. Editing assays: Each mutant was evaluated in an adenosine triphosphatase assay that measured tRNA-dependent editing by following the hydrolysis of ATP in the presence of l -valine and tRNA Ile . This type of assay does not distinguish between the hydrolysis of Val-AMP or the hydrolysis of Val-tRNA Ile . Final concentrations in each reaction mixture were 3 μM enzyme 4.6 μM tRNA Ile 140 mM tris (pH 7.5) 3 mM ATP (labeled with P 32 at the gamma position 12.5 μCi/ml) 0.5 mM CaCl 2 10 mM l -valine 9.3 mM MgCl 2 and pyrophosphatase (5 μg/ml). Each time point was quenched in 1.25 ml of a solution of 2 mM sodium pyrophosphate 8% activated charcoal 1.4% perchloric acid and 0.4% hydrochloric acid. After 10 min at room temperature the charcoal was pelleted by centrifugation and the phosphate content of 500 μl of each supernatant was quantitated by scintillation.
  20. 10.1126/science.276.5316.1250
  21. E. Schmidt and P. Schimmel unpublished data.
  22. S. F. Sneddon, D. J. Tobias, Biochemistry 31, 2842 (1992); S. E. Jackson, M. Moracci, N. Masry, C. M. Johnson, A. R. Fersht, ibid 32, 11259 (1993); Takano K., et al., J. Mol. Biol. 254, 62 (1995). (10.1006/jmbi.1995.0599) / J. Mol. Biol. by Takano K. (1995)
  23. Zhang Z. Y., Fasco M. J., Huang L., Guengerich F. P., Kaminsky L. S., Cancer Res.56, 3926 (1996). / Cancer Res. by Zhang Z. Y. (1996)
  24. Farrash M. A., et al., J. Virol.68, 233 (1994). (10.1128/jvi.68.1.233-239.1994) / J. Virol. by Farrash M. A. (1994)
  25. 10.1016/S0076-6879(97)76066-X
  26. CCP4: Collaborative Computational Project No 4 Acta Crystallogr. D 50 760 (1994). (10.1107/S0907444994003112)
  27. Abrahams J. P., Leslie A. G. W., ibid52, 30 (1996). / ibid by Abrahams J. P. (1996)
  28. 10.1107/S0108767390010224
  29. A. T. Brünger X-PLOR: A System for X-Ray Crystallography and NMR (Yale Univ. Press New Haven CT 1992).
  30. 10.1107/S0021889892009944
  31. Perona J. J., et al., Proc. Natl. Acad. Sci. U.S.A.88, 2903 (1991). (10.1073/pnas.88.7.2903) / Proc. Natl. Acad. Sci. U.S.A. by Perona J. J. (1991)
  32. We thank M. Nakasako N. Kamiya M. Yamamoto H. Iwasaki T. Ueki and Y. Maeda for data collection and helpful discussions; N. Watanabe and N. Sakabe for help with synchrotron data collection at the Tsukuba Photon Factory; and K. Nagai and A. Murzin for helpful comments. Supported in part by Grants-in-Aid for Scientific Research on Priority Areas (04272103 and 09278102) to S.Y. from the Ministry of Education Science Culture and Sports of Japan and by grant GM15539 to P.S. and T.H. from NIH. T.H. is an NIH Postdoctoral Fellow. The coordinates for T. thermophilus IleRS have been deposited in the Brookhaven Protein Data Base (1ILE).
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:37 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:21 a.m.)
Indexed 2 months, 2 weeks ago (June 10, 2025, 1:48 p.m.)
Issued 27 years, 4 months ago (April 24, 1998)
Published 27 years, 4 months ago (April 24, 1998)
Published Print 27 years, 4 months ago (April 24, 1998)
Funders 0

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@article{Nureki_1998, title={Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate}, volume={280}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.280.5363.578}, DOI={10.1126/science.280.5363.578}, number={5363}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Nureki, Osamu and Vassylyev, Dmitry G. and Tateno, Masaru and Shimada, Atsushi and Nakama, Takashi and Fukai, Shuya and Konno, Mitiko and Hendrickson, Tamara L. and Schimmel, Paul and Yokoyama, Shigeyuki}, year={1998}, month=apr, pages={578–582} }