DIRECTED MUTAGENESIS SHOWS THAT THE PRECEDING REGION OF THE CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE REGULATORY SEQUENCE IS THE THIOREDOXIN DOCKING SITE

The alignment of the six higher plant photosynthetic fructose-1,6-bisp hosphatases (FBPases) so far sequenced shows a lack of homology in the region which just precedes the cluster engaged in light modulation. E arlier experiments suggested that this region is the docking point in FBPase-thioredoxin (Trx) binding, and could be responsible for the int erspecific differences in the enzyme-Trx interaction and Trx ability f or FBPase activation. Using a pea chloroplast FBPase-coding cDNA, we h ave prepared two chimeric clones for FBPase. One of them (pDELFBP) sho ws a deletion of the 17 amino acids (Leu154 to Glu170) coding sequence , whereas in the second (pPFBPW) the above sequence was substituted by the corresponding one of the wheat enzyme. After Escherichia coli ove rexpression in pET-3d and later purification, both modified FBPases sh owed FBPase activity when determined under non-reducing conditions. Ho wever, only DELFBP lost the Trx f modulatory effect, indicating the im portant role played by this fragment in FBPase-Trx interaction and act ivity. Under these conditions the substituted PFBPW enzyme retains FBP ase activity, even though clearly diminished. Superose 12 filtration e xperiments after preincubating the wild-type and modified FBPases with Trx f, showed the existence of an enzyme-Trx f binding with the wild- type and the substituted PFBPW, but not with the deleted DELFBP protei n. Similarly, gradient PAGE under native conditions, followed by Weste rn blot and developing with FBPase and Trx f antibodies, indicated the existence of such a binding between the wild-type and PFBPW, on the o ne hand, and both Trxs f and m, on the other, although never with the deleted DELFBP enzyme. These results show the central role played by t he regulatory site preceding fragment of chloroplast FBPase in its bin ding with Trx. Computer-aided tridimensional models for the wild-type and modified FBPases are proposed. (C) 1997 Academic Press Limited.