title: Purification and biochemical properties of phytochromobilin synthase from etiolated oat seedlings.
abstract: Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (P Phi B) for photoactivity. In planta, biliverdin IX alpha (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme P Phi B synthase to yield 3Z-P Phi B. Here, we describe the >50,000-fold purification of P Phi B synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, P Phi B synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s(-1), which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat P Phi B synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of P Phi B synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A K(m) for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 microM. P Phi B synthase has a high affinity for its bilin substrate, with a sub-micromolar K(m) for BV.
authors.affiliation: Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California 95616, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: McDowell
authors.forename: J C
authors.initials: JC
authors.lastname: Lagarias
mesh_terms: D018554:Avena / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D001664:Biliverdine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002734:Chlorophyll; D005288:Ferredoxins / Q000378:metabolism; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010834:Phytochrome / Q000737:chemistry; D018520:Plant Shoots; D018087:Plastids / Q000737:chemistry
chemical_list: D005288:Ferredoxins; D010834:Phytochrome; D002734:Chlorophyll; C075811:phytochromobilin; D010088:Oxidoreductases; C071703:phytochromobilin synthase; D001664:Biliverdine