Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures
1999
Prior, A. | Uhrig, J.F. | Heins, L. | Wiesmann, A. | Lillig, C.H. | Stoltze, C. | Soll, J. | Schwenn, J.D.
A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784). The open reading frame of 1392 bp (termed par) encoded for a protein (Mr = 51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa. The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies. To investigate the catalytical function of the different domains deleted par proteins were purified. Par delta 1 lacking the transit sequence liberated sulfite from APS (K(m) 2.5 +/- 0.23 micromolar) in vitro with glutathione (K(m) 3 +/- 0.64 mM) as reductant (V(max) 2.6 +/- 0.14 U mg-1, molecular activity 126 min-1). Par delta 2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (K(m) 15.3 +/- 1.27 micromolar, V(max) 0.6 +/- 0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. Par delta 1 (35.4%) and par delta 2 (21.8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for par delta 1.
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