Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures

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 pal) encoded for a protein (M-r = 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 approximate to 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 mu M) 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 mu M, 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. (C) 1999 Elsevier Science B.V. All rights reserved.