Solution structure of 3-oxo-Δ5-steroid isomerase

Science

Volumn 276, Issue 5311, 1997, Pages 415-418

  Wu, Zheng Rong ^a   Ebrahimian, Soheila ^a   Zawrotny, Michael E ^a   Thornburg, Lora D ^a   Perez Alvarado, Gabriela C ^a   Brothers, Paul ^a   Pollack, Ralph M ^a,b   Summers, Michael F ^a  

^a UNIVERSITY OF MARYLAND BALTIMORE COUNTY   (United States)

^b UNIVERSITY OF MARYLAND BIOTECHNOLOGY INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ASPARTIC ACID; PRASTERONE; STEROID DELTA ISOMERASE; TYROSINE;

ALPHA HELIX; ARTICLE; CATALYSIS; CONTROLLED STUDY; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME STRUCTURE; HYDROGEN BOND; HYDROPHOBICITY; NONHUMAN; NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY; NUCLEAR OVERHAUSER EFFECT; PH; PRIORITY JOURNAL; PROTEIN AGGREGATION; PROTEIN SECONDARY STRUCTURE;

AMINO ACID SEQUENCE; ANDROSTENEDIONE; BINDING SITES; DIMERIZATION; ESTRADIOL; HYDROGEN BONDING; MAGNETIC RESONANCE SPECTROSCOPY; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MUTAGENESIS, SITE-DIRECTED; PROTEIN CONFORMATION; PROTEIN STRUCTURE, SECONDARY; SOLUTIONS; STEROID ISOMERASES;

EID: 0000346064     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5311.415     Document Type: Article

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27. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
28. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
29. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
30. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
31. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
32. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
33. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
34. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
35. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
36. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
37. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
38. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
39. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
40. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
41. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
42. 13C NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87 (1994)].
43. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
44. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
45. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
46. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
47. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
48. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
49. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
50. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
51. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
52. 13Cα chemical shift indices [S. Spera and A. Bax, J. Am. Chem. Soc., 113, 5490 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 222, 311 (1991); D. S. Wishart, B. D. Sykes, F. M. Richards, Biochemistry 31, 1647 (1992); D. S. Wishart and B. D. Sykes, J. Biomol. NMR 4, 171 (1994)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were only employed for the stronger of the two cross peaks). Color figures were generated with Midas-plus [T. E. Ferrin, C. C. Huang, L. E. Jarvis, R. Langridge, J. Mol. Graph. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], and Raster-30 p. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
53. A. T. Brünger, X-PLOR: A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, ed. 3.1, 1993).
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55. D. C. Hawkinson and R. M. Pollack, ibid. 32, 694 (1993).
56. R. D. Gandour, Bioorg. Chem. 10, 169 (1981).
57. Single letter abbreviations for the amino acids are: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
58. Oligonucleotide-directed mutagenesis was performed according to the method of Eckstein and coworkers [J. R. Sayers, C. Krekel, F. Eckstein, BioTechniques 13, 592 (1992)]. The D99A protein was expressed in DH5α E. coli (grown in LB medium and induced with isopropyl-β-thiogalactopyranoside), and purified as described above (79). The resulting protein was homogeneous, on the basis of SDS-PAGE. Steady-state kinetic constants for the reaction of D99A with 5-androstene-3, 17-dione (1) were determined in 34 mM phosphate, pH 7.0, 3.3% methanol as previously described for wild-type enzyme [R. M. Pollack, S. Bantia, P. L. Bounds, B. M. Koffman, Biochemistry 25, 1905 (1986)].
59. Oligonucleotide-directed mutagenesis was performed according to the method of Eckstein and coworkers [J. R. Sayers, C. Krekel, F. Eckstein, BioTechniques 13, 592 (1992)]. The D99A protein was expressed in DH5α E. coli (grown in LB medium and induced with isopropyl-β-thiogalactopyranoside), and purified as described above (79). The resulting protein was homogeneous, on the basis of SDS-PAGE. Steady-state kinetic constants for the reaction of D99A with 5-androstene-3, 17-dione (1) were determined in 34 mM phosphate, pH 7.0, 3.3% methanol as previously described for wild-type enzyme [R. M. Pollack, S. Bantia, P. L. Bounds, B. M. Koffman, Biochemistry 25, 1905 (1986)].
60. i of > 100 μM.
61. i of > 100 μM.
62. A high pKa (>9) has been observed for a buried aspartic acid in reduced thioredoxin [N. A. Wilson. E. Barbar, K. Langsetmo, J. A. Fuchs, C. Woodward, Biochemistry 34, 8931 (1995)].
63. 99 by Ala. Further experiments are thus required to identify the origin of the downfield-shifted signal.
64. S. Shan and D. Herschlag, J. Am. Chem. Soc. 118, 5515 (1996).
65. Supported by NIH grants GM38155 and GM49082. We thank M. C. Betlach for supplying us with the E. coli strain NCM933, and M. R. Starich for technical assistance and for collecting some of the NMR data. Coordinates and NMR restraints have been deposited in the Brookhaven Protein Data Bank with accession numbers 1ISK and R1ISKMR, respectively.