A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus.
In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.
Nomata
Jiro
J
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Mizoguchi
Tadashi
T
Tamiaki
Hitoshi
H
Fujita
Yuichi
Y
D000255:Adenosine Triphosphate / Q000737:chemistry; D001429:Bacteriochlorophylls / Q000096:biosynthesis*; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004227:Dithionite / Q000737:chemistry; D008954:Models, Biological; D008956:Models, Chemical; D009591:Nitrogenase / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000378:metabolism*
D001429:Bacteriochlorophylls; D017931:DNA Primers; D010455:Peptides; D004227:Dithionite; D000255:Adenosine Triphosphate; D010105:Oxygenases; C115272:chlorophyll a oxygenase; D009591:Nitrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C511363:chlorophyllide a reductase, Rhodobacter capsulatus; D003545:Cysteine