Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods
1996 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 317, no 2, 549-555 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
1996. Vol. 317, no 2, 549-555 p.
IdentifiersURN: urn:nbn:se:umu:diva-44692ISI: A1996UZ33400030OAI: oai:DiVA.org:umu-44692DiVA: diva2:428162