Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga 1

FEBS Letters

Wastl, Jürgen¹  Sticht, Heinrich²  Hoffmann, Silke²  Rösch, Paul²  Maier, Uwe-G.¹ 

[1] Cell Biology and Applied Botany, Philipps-University Marburg, Karl-von-Frisch-Strasse, D-35032 Marburg, Germany;Lehrstuhl für Biopolymere, Universität Bayreuth, Universitätsstr. 30, D-95440 Bayreuth, Germany

关键词: Rubredoxin;    Cryptomonad;    Nucleomorph;    Transit peptide;    Iron binding site;    DEAE;    diethylaminoethyl;    ϵ;    extinction coefficient;    IPTG;    isopropyl-β-D-thiogalactopyranoside;    MALDI;    matrix-assisted laser desorption ionization;    NMR;    nuclear magnetic resonance;    OD600;    optical density measured at 600 nm;    PAGE;    polyacrylamide gel electrophoresis;    rd;    rubredoxin;    Tris;    tris(hydroxymethyl)aminoethane;   

DOI  :  10.1016/S0014-5793(00)01399-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH₂-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary 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-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.

【 授权许可】

Unknown