The CXXC motif: crystal structure of an active-site variant of Escherichiacoli thioredoxin

The 2.2 Angstrom crystalline structure of an oxidized active-site variant o f Escherichia coli thioredoxin (Trx) has been solved. Trx is a 12 kDa enzym e which catalyzes the oxidation of dithiols and the reduction and isomeriza tion of disulfides in other proteins. Its active site contains the common s tructural motif CXXC, Protein-disulfide isomerase (PDI), a 57 kDa homolog o f Trx, contains four Trx-like domains. The three-dimensional structure of P DI is unknown. PDI-deficient Saccharomyces cerevisiae are inviable. An acti ve-site variant of Trx which complements PDI-deficient yeast has the active -site sequence Cys32-Val33-Trp34-Cys35 (CVWC). The reduction potential of o xidized CVWC Trx (E degrees' = -0.230 V) is altered significantly from that of the wild-type enzyme (E degrees' = -0.270 V). However, the structure of the oxidized CVWC enzyme is almost identical to that of wild-type Tx. The addition of valine and tryptophan in the active site is likely to increase the reduction potential, largely by decreasing the pK(a) of the Cys32 thiol in the reduced enzyme. Unlike in wild-type Trx, significant protein-protei n contacts occur in the crystal. Protein molecules related by a crystallogr aphic twofold axis form a dimer in the crystal. The dimer forms as an exten sion of the twisted mixed beta-sheet which composes the backbone of each Tr x structure.