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
B. Bednarski et al., 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 BIOCH, 268(12), 2001, pp. 3538-3544
GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase
and D-proline reductase, respectively, that are processed post-translationa
lly 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 py
ruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli
and conditions were developed for in vitro processing. GrdE could be expre
ssed as full-size protein, whereas PrdA had to be truncated N-terminally to
achieve successful over-expression. Both proproteins were cleaved at the i
n 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 G
rdE was observed with a half-time of approximate to 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 extende
d half-times. However, the Cys242 --> Ala mutant protein was not cleaved in
dicating a pivotal role of the thiol group of cysteine or hydroxyl group of
serine and threonine during the processing of pyruvoyl group-dependent red
uctases.