Biochemical properties of the smallest PDI like protein of Arabidopsis thaliana

Protein disulfide isomerases (PDI) assist in vivo protein folding by helping newly translated polypeptide chains to form native disulfide bonds. PDI structure consists in two tandem repeats of thioredoxin domains, a-b-b-'a', in which a and a' domains are catalytically active. This enzyme is the founding member of a family of proteins that vary in length and domain arrangement, but share the common structural feature of having at least one domain with a thioredoxin-like fold (1). In A. thaliana there are at least 12 putative PDI-like enzymes (PDIL) (2). Among them AtPDIL 5-1 is the only single domain member. The human counterpart (HsERp18) has been biochemical characterized as an ER-resident disulfide oxidase (3). Here we present a preliminary biochemical characterization of AtPDIL 5-1. De novo in silico structural analysis suggests that AtPDIL 5-1 maintains the Rossman folding typical of thioredoxin-like proteins. In order to assess the in vitro activity of the protein, AtPDIL 5-1 was over-expressed in E. coli. For comparison we also produced a recombinant version of a canonical PDI (AtPDIL 1-1) and a truncated form of it, consisting only in the a domain (PDIa). The proteins were expressed and purified via Ni-affinity chromatography and tested for in vitro thioredoxin-like activities. When compared to the recombinant full length and truncated PDIs, AtPDIL 5-1 is significantly less active in both the reduction and the oxidation assay. These preliminary data suggest that HsERp18 and AtPDIL 5-1 does not share the same catalytic activity thus justifying further studies on the biochemical properties of this protein.