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

Biochim Biophys Acta. 1999 Feb 10;1430(1):25-38. doi: 10.1016/s0167-4838(98)00266-0.

Abstract

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. ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1). ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15. 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%) and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for parDelta1.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cells, Cultured
  • DNA, Chloroplast / isolation & purification*
  • DNA, Chloroplast / metabolism
  • DNA, Complementary / isolation & purification*
  • DNA, Complementary / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Oxidoreductases Acting on Sulfur Group Donors*
  • Sequence Alignment

Substances

  • DNA, Chloroplast
  • DNA, Complementary
  • Oxidoreductases
  • Oxidoreductases Acting on Sulfur Group Donors
  • adenylylsulfate reductase

Associated data

  • GENBANK/U63784