Chorismate synthase (EC 4.6.1.4) catalyses the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) into chorismate, and requires reduced FMN as a cofactor. The enzyme can bind first oxidized FMN and then EPSP to form a stable ternary complex which does not undergo turnover. This complex can be considered to be a model of the ternary complex between enzyme, EPSP and reduced FMN immediately before catalysis commences. It is shown that the binding of oxidized FMN and EPSP to chorismate synthase affects the properties and structure of the protein. Changes in small-angle X-ray scattering data, decreased susceptibility to tryptic digestion and altered Fourier-transform (FT)-IR spectra provide the first strong evidence for major structural changes in the protein. The tetrameric enzyme undergoes correlated screw movements leading to a more overall compact shape, with no change in oligomerization state. The changes in the FT-IR spectrum appear to reflect changes in the environment of the secondary-structural elements rather than alterations in their distribution, because the far-UV CD spectrum changes very little. Changes in the mobility of the protein during non-denaturing PAGE indicate that the ternary complex may exhibit less conformational flexibility than the apoprotein. Increased enzyme solubility and decreased tryptophan fluorescence are discussed in the light of the observed structural changes. The secondary structure of the enzyme was investigated using far-UV CD spectroscopy, and the tertiary structure was predicted to be an α–β-barrel using discrete state-space modelling.
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Research Article|
October 15 1998
Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding
Peter MACHEROUX;
*ETH-Zürich, Institute of Plant Sciences, Universitätstr. 2, CH-8092 Zürich, Switzerland
†Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, U.K.
2To whom correspondence should be addressed (e-mail roger.thorneley@bbsrc.ac.uk).
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Ernst SCHÖNBRUNN;
Ernst SCHÖNBRUNN
*ETH-Zürich, Institute of Plant Sciences, Universitätstr. 2, CH-8092 Zürich, Switzerland
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Dmitri I. SVERGUN;
Dmitri I. SVERGUN
‡EMBL, Hamburg Outstation, Notkestrasse 85, D-22603, Hamburg, Germany
§Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
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Vladimir V. VOLKOV;
Vladimir V. VOLKOV
§Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
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Michel H. J. KOCH;
Michel H. J. KOCH
‡EMBL, Hamburg Outstation, Notkestrasse 85, D-22603, Hamburg, Germany
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Stephen BORNEMANN;
Stephen BORNEMANN
†Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, U.K.
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Roger N. F. THORNELEY
Roger N. F. THORNELEY
2
†Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, U.K.
2To whom correspondence should be addressed (e-mail roger.thorneley@bbsrc.ac.uk).
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Publisher: Portland Press Ltd
Received:
June 18 1998
Revision Received:
July 22 1998
Accepted:
August 13 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 335 (2): 319–327.
Article history
Received:
June 18 1998
Revision Received:
July 22 1998
Accepted:
August 13 1998
Citation
Peter MACHEROUX, Ernst SCHÖNBRUNN, Dmitri I. SVERGUN, Vladimir V. VOLKOV, Michel H. J. KOCH, Stephen BORNEMANN, Roger N. F. THORNELEY; Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding. Biochem J 15 October 1998; 335 (2): 319–327. doi: https://doi.org/10.1042/bj3350319
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