title: Plant Protochlorophyllide Oxidoreductases A and B: CATALYTIC EFFICIENCY AND INITIAL REACTION STEPS.
abstract: The enzyme protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) has a key role in plant development. It catalyzes one of the later steps in chlorophyll synthesis, the light-induced reduction of protochlorophyllide (PChlide) into chlorophyllide (Chlide) in the presence of NADPH. Two isozymes of plant POR, POR A and POR B from barley, which differ in their function during plant life, are compared with respect to their substrate binding affinity, catalytic efficiency, and catalytic mechanism. POR B as compared with POR A shows an 5-fold higher binding affinity for PChlide and an about 6-fold higher catalytic efficiency measured as kcat/Km. Based on the reaction intermediates, which can be trapped at low temperatures the same reaction mechanism operates in both POR A and POR B. In contrast to results reported for POR enzymes from cyanobacteria, the initial light-driven step, which occurs at temperatures below 180 K already involves the full chemistry of the photoreduction and yields the reaction product, Chlide, in an enzyme-bound form. The subsequent dark reactions, which include cofactor (NADP(+)) release and cofactor (NADPH) rebinding, show different temperature dependences for POR A and POR B and suggest a higher conformational flexibility of POR B in the surrounding active center. Both the higher substrate binding affinity and well adapted enzyme dynamics are held responsible for the increased catalytic activity of POR B as compared with POR A.
authors.affiliation: Department of Biochemistry and Biophysics, and the Department of Physical Chemistry, Jena D-07743, Germany; Leibniz Institute of Photonic Technology, Jena D-07743, Germany.
authors.forename: Alessio
authors.initials: A
authors.lastname: Garrone
authors.affiliation: Department of Biochemistry and Biophysics, and the Department of Physical Chemistry, Jena D-07743, Germany.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Archipowa
authors.affiliation: Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena D-07743, Germany; Friedrich Schiller University of Jena, Jena D-07743, Germany.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Zipfel
authors.affiliation: Department of Biochemistry and Biophysics, and the Department of Physical Chemistry, Jena D-07743, Germany; Friedrich Schiller University of Jena, Jena D-07743, Germany. Electronic address: Gudrun.Hermann@uni-jena.de.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Hermann
authors.affiliation: Department of Biochemistry and Biophysics, and the Department of Physical Chemistry, Jena D-07743, Germany; Leibniz Institute of Photonic Technology, Jena D-07743, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dietzek
mesh_terms: D055162:Biocatalysis; D002734:Chlorophyll / Q000096:biosynthesis; D000458:Cyanobacteria / Q000201:enzymology / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D011521:Protochlorophyllide / Q000378:metabolism*
chemical_list: D002734:Chlorophyll; D011521:Protochlorophyllide; D010088:Oxidoreductases
keywords: NADPH:protochlorophyllide oxidoreductase; chlorophyll; chlorophyll biosynthesis; enzyme kinetics; low temperature absorption spectroscopy; plant biochemistry; reductase; ultraviolet-visible spectroscopy (UV-Vis spectroscopy)