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
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Medientyp:
E-Artikel
Titel:
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
:
Formation of a pyruvoyl group from a cysteine residue
Beteiligte:
Bednarski, Brit;
Andreesen, Jan R.;
Pich, Andreas
Beschreibung:
<jats:p>GrdE and PrdA of <jats:italic>Clostridium sticklandii</jats:italic> are subunits of glycine reductase and <jats:sc>d</jats:sc>‐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 <jats:italic>Escherichia coli</jats:italic> and conditions were developed for <jats:italic>in vitro</jats:italic> 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 <jats:italic>in vivo</jats:italic> observed cleavage site after addition of 200 m<jats:sc>m</jats:sc> NaBH<jats:sub>4</jats:sub> 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 ≈ 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.</jats:p>