Cloning of the gene encoding a protochlorophyllide reductase: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the cyanobacterium Plectonema boryanum.
Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase. We found an open reading frame (ORF322) in a 2,131-bp EcoRI fragment from the genomic DNA of the cyanobacterium Plectonema boryanum. Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the por gene encoding LPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolated by targeted mutagenesis to investigate the physiological importance of LPOR. YFP12 grew as well as wild type under low light conditions (10-25 muE m-2 s-1). However, its growth was significantly retarded as a result of a significant decrease in its Chl content under higher light conditions (85-130 muE m-2 s-1). Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 muE m-2 s-1). In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity. From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chl synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130 muE m-2 s-1.
Fujita
Y
Y
Division of Enzymology, Osaka University, Japan.
Takagi
H
H
Hase
T
T
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D003624:Darkness; D004926:Escherichia coli; D005798:Genes, Bacterial; D008027:Light; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010944:Plants / Q000201:enzymology; D011521:Protochlorophyllide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D011521:Protochlorophyllide; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase