Reconstitution of light-independent protochlorophyllide reductase from purified bchl and BchN-BchB subunits. In vitro confirmation of nitrogenase-like features of a bacteriochlorophyll biosynthesis enzyme.
Protochlorophyllide reductase catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls and bacteriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photosynthetic bacteria to green (form chlorophyll) in the dark. Genetic and sequence analyses have indicated that dark protochlorophyllide reductase consists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen. However, unlike the well characterized features of nitrogenase, there has been no previous biochemical characterization of dark protochlorophyllide reductase. In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R. capsulatus as S tag fusion proteins that facilitated affinity purification. The third subunit (BchB) was co-purified with the BchN protein indicating that BchN and BchB proteins form a tight complex. Dark protochlorophyllide reductase activity was shown to be dependent on the presence of all three subunits, ATP, and the reductant dithionite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed.
Fujita
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Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Bauer
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CE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001429:Bacteriochlorophylls / Q000096:biosynthesis*; D001483:Base Sequence; D003624:Darkness; D004227:Dithionite / Q000378:metabolism; D008969:Molecular Sequence Data; D009591:Nitrogenase; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology*
D001429:Bacteriochlorophylls; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D004227:Dithionite; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase