Identification and characterization of a eukaryotically encoded rubredoxinin a cryptomonad alga

We have identified an open reading frame,with homology to prokaryotic rubre doxins (rds) on a nuclcomorph chromosome of the cryptomonad alga Guillardia theta, cDNA analysis let us propose that the rd preprotein has an NH2-term inal extension that functions as a transit peptide for import into the plas tid, Compared to rds found in non-photosynthetic prokaryotes or found in ba cteria that exhibit an anoxigenic photosynthesis apparatus, nuclcomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical I'd in the nl Arabidopsis genome, This extension can be divided into a putative membr ane anchor and a stretch of about 20 amino acids with unknown function link ing the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mi xture of iron-containing holorubredoxin and zinc-substituted protein, Preli minary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-l cl suggests the presence of a st able tertiary fold similar to that of other rd structures determined previo usly. (C) 2000 Federation of European Biochemical Societies.