In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essen
tial catalyst for disulfide bond formation in the bacterial periplasm share
s with other oxidoreductases of the thioredoxin family a cis-proline in pro
ximity of the active site residues. In the variant DsbA(P151A), this residu
e has been changed to an alanine, an almost isosteric residue which is not
disposed to adopt the cis conformation. The substitution strongly destabili
zed the structure of DsbA, as determined by the decrease in the free energy
of folding. The pK(a) of the thiol of Cys30 was only marginally decreased.
Although in vivo the variant appeared to be correctly oxidized, it exhibit
ed an activity less than half that of the wild-type enzyme with respect to
the folding of alkaline phosphatase, used as a reporter of the disulfide bo
nd formation in the periplasm. DsbA(P151A) crystallized in a different crys
tal form from the wild-type protein, in space group P2(1) with six molecule
s in the asymmetric unit. Its X-ray structure was determined to 2.8 Angstro
m resolution. The most significant conformational changes occurred at the a
ctive site. The loop 149-152 adopted a new backbone conformation with Ala15
1 in a trans conformation. This rearrangement resulted in the loss of van d
er Waals interactions between this loop and the disulfide bond. His32 from
the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the
asymmetric unit a gauche-conformation not observed in the wild-type protein
. The X-ray structure and folding studies on DsbA(P151A) were consistent wi
th the cis-proline playing a major role in the stabilization of the protein
. A role for the positioning of the substrate is discussed. These important
properties for the enzyme function might explain the conservation of this
residue in DsbA and related proteins possessing the thioredoxin fold.