POR structural domains important for the enzyme activity in <Emphasis Type="Italic">R. capsulatus</Emphasis> complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. Abbreviations: BChl – bacteriochlorophyll; BPheo – bacteriopheophytin; Chlide – chlorophyllide; CP – Chlorophyll–protein; LH – light-harvesting complex; Mg-PP IX – magnesium protoporphyrin IX; PAGE – polyacrylamide gel electrophoresis; PChlide – protochlorophyllide; POR – NADPH:protochlorophyllide oxidoreductase; RC – photosynthetic reaction center; SDS – sodium dodecyl sulfate; WT – wild type