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.
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.
Bednarski
B
B
Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
Andreesen
J R
JR
Pich
A
A
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003013:Clostridium / Q000201:enzymology* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009097:Multienzyme Complexes; D010446:Peptide Fragments; C022452:D-proline reductase (dithiol); D000594:Amino Acid Oxidoreductases; C020815:glycine reductase; D003545:Cysteine