title: Functional evaluation of a nitrogenase-like protochlorophyllide reductase encoded by the chloroplast DNA of Physcomitrella patens in the cyanobacterium Leptolyngbya boryana.
abstract: Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of the two components, L-protein (a ChlL dimer) and NB-protein (a ChlN-ChlB heterotetramer), to catalyze Pchlide reduction in Chl biosynthesis. While nitrogenase is distributed only among certain prokaryotes, the probable structural genes for DPOR are encoded by chloroplast DNA in lower plants. Here we show functional evaluation of DPOR encoded by chloroplast DNA in a moss Physcomitrella patens by the complementation analysis of the cyanobacterium Leptolyngbya boryana and the heterologous reconstitution of the moss L-protein and the cyanobacterial NB-protein. Two shuttle vectors to overexpress chlL and chlN-chlB from P. patens were introduced into the cyanobacterial chlL- and chlB-lacking mutants, respectively. Both transformants restored the ability to perform Chl biosynthesis in the dark, indicating that the chloroplast-encoded DPOR components form an active complex with the cyanobacterial components. The L-protein of P. patens was purified from the cyanobacterial transformant, and DPOR activity was reconstituted in a heterologous combination with the cyanobacterial NB-protein. The specific activity of the L-protein from P. patens was determined to be 118 nmol min(-1) mg (-1), which is even higher than that of the cyanobacterial L-protein (76 nmol min(-1) mg (-1)). Upon exposure to air, the activity of the L-protein from P. patens decayed with a half-life of 30 s, which was eight times faster than that of the cyanobacterial L-protein (240 s). These results suggested that the chloroplast-encoded L-protein functions as efficiently as the cyanobacterial L-protein but is more oxygen labile than the cyanobacterial L-protein.
authors.affiliation: School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Haruki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Shohei
authors.initials: S
authors.lastname: Kurumiya
authors.forename: Rie
authors.initials: R
authors.lastname: Ohashi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Fujita
mesh_terms: D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics; D002734:Chlorophyll / Q000096:biosynthesis; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D018742:DNA, Chloroplast / Q000235:genetics*; D005816:Genetic Complementation Test; D009154:Mutation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018742:DNA, Chloroplast; D010940:Plant Proteins; D002734:Chlorophyll; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D010100:Oxygen