Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase

Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9287-91. doi: 10.1073/pnas.93.17.9287.


delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde Oxidoreductases / chemistry
  • Aldehyde Oxidoreductases / genetics
  • Aldehyde Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Aminolevulinic Acid / metabolism
  • Base Sequence
  • Cytochromes / metabolism
  • Escherichia coli / genetics
  • Glutamates / metabolism
  • Glutamic Acid / metabolism
  • Glutathione Transferase / biosynthesis
  • Glutathione Transferase / chemistry
  • Glutathione Transferase / genetics
  • Heme / metabolism
  • Hemin / pharmacology
  • Hordeum / enzymology*
  • Hordeum / genetics
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Spectrophotometry


  • Cytochromes
  • Glutamates
  • Recombinant Fusion Proteins
  • Glutamic Acid
  • Heme
  • glutamate-1-semialdehyde
  • Hemin
  • Aminolevulinic Acid
  • Aldehyde Oxidoreductases
  • glutamyl tRNA reductase
  • Glutathione Transferase