In Vitro Processing of the Proproteins GrdE of Protein B of Glycine Reductase and PrdA of D-proline Reductase From Clostridium Sticklandii: Formation of a Pyruvoyl Group From a Cysteine Residue

Eur J Biochem. 2001 Jun;268(12):3538-44. doi: 10.1046/j.1432-1327.2001.02257.x.

Abstract

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of approximately 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Oxidoreductases / chemistry
  • Amino Acid Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Catalytic Domain
  • Clostridium / enzymology*
  • Clostridium / metabolism
  • Cysteine / chemistry
  • DNA Primers
  • Hydrolysis
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / metabolism*
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Protein Processing, Post-Translational*
  • Sequence Homology, Amino Acid

Substances

  • DNA Primers
  • Multienzyme Complexes
  • Peptide Fragments
  • D-proline reductase (dithiol)
  • Amino Acid Oxidoreductases
  • glycine reductase
  • Cysteine