Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods.
To study the secondary structure of the enzyme NADPH: protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly-(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivum L.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, alpha-helix, beta-sheet, turn and random coil. The secondary structure composition was estimated to be 33% alpha-helix, 19% beta-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a beta-sheet or an alpha-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring.
Birve
S J
SJ
Department of Plant Physiology, Umeå University, Sweden.
Selstam
E
E
Johansson
L B
LB
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D005544:Forecasting / Q000379:methods; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010940:Plant Proteins / Q000737:chemistry*; D018087:Plastids / Q000201:enzymology; D017433:Protein Structure, Secondary; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014908:Triticum
D008565:Membrane Proteins; D010940:Plant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase