Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein dis ulfide reductases in living cells. Chloroplasts contain two distinct thiore doxins (f and m) with different phylogenetic origin. Both act as enzyme reg ulatory proteins but have different specificities towards target enzymes. T hioredoxin f (Trx f), which shares only low sequence identity with thioredo xin m (Trx m) and with all other known thioredoxins, activates enzymes of t he Calvin cycle and other photosynthetic processes. Trx m shows high sequen ce similarity with bacterial thioredoxins and activates other chloroplast e nzymes. The here described structural studies of the two chloroplast thiore doxins were carried out in order to gain insight into the structure/functio n relationships of these proteins. Crystal structures were determined for o xidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncate d form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m ( at 2.1 and 2.3 Angstrom resolution, respectively). Whereas thioredoxin f cr ystallized as a monomer, both truncated thioredoxin f and thioredoxin m cry stallized as non-covalent dimers. The structures of thioredoxins f and m ex hibit the typical thioredoxin fold consisting of a central twisted five-str anded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains a n additional alpha-helix at the N terminus and an exposed third cysteine cl ose to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribut e to the specificity of thioredoxin f is an inherent flexibility of its act ive site, which has expressed itself crystallographically in two different crystal forms. (C) 2000 Academic Press.