Purification and biochemical properties of phytochromobilin synthase from etiolated oat seedlings.
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.
McDowell
M T
MT
Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California 95616, USA.
Lagarias
J C
JC
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
D005288:Ferredoxins; D010834:Phytochrome; D002734:Chlorophyll; C075811:phytochromobilin; D010088:Oxidoreductases; C071703:phytochromobilin synthase; D001664:Biliverdine