A homolog of old yellow enzyme in tomato. Spectral properties and substrate specificity of the recombinant protein.
A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) from Arabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of the LeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration with para-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR.
Strassner
J
J
Institute of Plant Sciences, Federal Institute of Technology Zürich, Universitätstrasse 2, CH-8092 Zürich, Switzerland.
Fürholz
A
A
Macheroux
P
P
Amrhein
N
N
Schaller
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D018551:Solanum lycopersicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010636:Phenols / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D018076:DNA, Complementary; D005415:Flavins; D010636:Phenols; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C106360:12-oxophytodienoate reductase; D009252:NADPH Dehydrogenase