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Plant Physiol, August 2001, Vol. 126, pp. 1546-1554
Purification and Biochemical Properties of Phytochromobilin
Synthase from Etiolated Oat Seedlings1
Michael T.
McDowell2 and
J. Clark
Lagarias*
Section of Molecular and Cellular Biology, University of
California, One Shields Avenue, Davis, California 95616
Plant phytochromes are dependent on the covalent attachment of the
linear tetrapyrrole chromophore phytochromobilin (P B) for
photoactivity. In planta, biliverdin IX (BV) is reduced by the
plastid-localized, ferredoxin (Fd)-dependent enzyme PB synthase to
yield 3Z-PB. Here, we describe the >50,000-fold purification of
PB synthase from etioplasts from dark-grown oat (Avena
sativa L. cv Garry) seedlings using traditional column
chromatography and preparative electrophoresis. Thus, PB 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 PB 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 PB synthase to reduce BV is dependent on reduced
2Fe-2S Fds. A Km for spinach
(Spinacea oleracea) Fd was determined to be 3 to 4 µM. PB synthase has a high affinity for its bilin
substrate, with a sub-micromolar Km for BV.
1
This work was supported by the U.S. Department
of Agriculture National Research Initiative Competitive Grants Program
(grant no. AMD-9801768 to J.C.L.).
2
Present address: Amersham Pharmacia Biotech, 654 Minnesota Street, San Francisco, CA 94107-0387.
*
Corresponding author, e-mail jclagarias{at}ucdavis.edu; fax
530-752-3085.
© 2001 American Society of Plant Physiologists
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