title: Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus.
abstract: Dark-operative protochlorophyllide oxidoreductase (DPOR) plays a crucial role in light-independent (bacterio)chlorophyll biosynthesis in most photosynthetic organisms. However, the biochemical properties of DPOR are still largely undefined. Here, we constructed an overexpression system of two separable components of DPOR, L-protein (BchL) and NB-protein (BchN-BchB), in the broad-host-range vector pJRD215 in Rhodobacter capsulatus. We established a stable DPOR assay system by mixing crude extracts from the two transconjugants under anaerobic conditions. Using this assay system, we demonstrated some basic properties of DPOR. The Km value for protochlorophyllide was 10.6 muM. Ferredoxin functioned as an electron donor to DPOR. Elution profiles in gel filtration chromatography indicated that L-protein and NB-protein are a homodimer [(BchL)(2)] and a heterotetramer [(BchN)(2)(BchB)(2)], respectively. These results provide a framework for the characterization of these components in detail, and further support a nitrogenase model of DPOR.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Nomata
authors.forename: Lee R
authors.initials: LR
authors.lastname: Swem
authors.forename: Carl E
authors.initials: CE
authors.lastname: Bauer
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Fujita
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D003227:Conjugation, Genetic; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D005288:Ferredoxins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase