Cloning and sequencing of protochlorophyllide reductase

Abstract

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a A-phage gtl cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase. p127 codes for more than 95 %o of the reductase protein.

Molecular aspects of plastid envelope biochemistry

“…This compares with a doublet of 35 and 37 kDa as the mature polypeptides in 6-day-old etiolated oats [109]. In our hands, the envelope polypeptide from mature spinach chloroplasts always migrates as a single polypeptide.…”

“…Protochlorophyllide reductase is coded for by nuclear DNA and is synthesized, in oat, as a 41-kDa precursor [109]. This compares with a doublet of 35 and 37 kDa as the mature polypeptides in 6-day-old etiolated oats [109].…”

“…In our hands, the envelope polypeptide from mature spinach chloroplasts always migrates as a single polypeptide. cDNA clones for protochlorophyllide reductase were used as a probe for investigating some aspects of protochlorophyllide reductase formation [109-11 11 and to determine its nucleotide sequence [109]. The protochlorophyllide reductase contain a relatively high content of basic amino acids, with arginine and lysine accounting for more than 12% of the total [109].…”

“…cDNA clones for protochlorophyllide reductase were used as a probe for investigating some aspects of protochlorophyllide reductase formation [109-11 11 and to determine its nucleotide sequence [109]. The protochlorophyllide reductase contain a relatively high content of basic amino acids, with arginine and lysine accounting for more than 12% of the total [109]. However, it is not yet known how many genes code for protochlorophyllide reductase and whether the presence of multiple genes could be responsible for the existence of the different isoenzymes [I 121 present in different membrane systems (outer envelope membrane, prolamellar body, etc.).…”

Genetic analyses of photopigment biosynthesis in eubacteria: a guiding light for algae and plants

“…Theoretically, it should also be possible to utilize the technology of reverse genetics to clone loci that code for enzyme subunits that catalyze chlorophyll biosynthesis reactions. However, only one enzyme involved in the Mg branch of the pathway, light-dependent NADPH:protochlorophyllide oxidoreductase, has been purified to a degree of homogeneity that has allowed the production of antibodies for use in cDNA expression library screening and subsequent cloning of the gene from plants (15,50). The difficulty in isolating these enzymes appears to stem, in part, from the low cellular levels of these enzymes coupled with their association with membrane fractions.…”