Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding.
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 alpha-beta-barrel using discrete state-space modelling.
ETH-Zürich, Institute of Plant Sciences, Universitätstr. 2, CH-8092 Zürich, Switzerland.
P
P
Macheroux
E
E
Schönbrunn
D I
DI
Svergun
V V
VV
Volkov
M H
MH
Koch
S
S
Bornemann
R N
RN
Thorneley
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan; D014466:Ultraviolet Rays
C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D005486:Flavin Mononucleotide; D014364:Tryptophan; D014357:Trypsin; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases