Crystal structure of the non-regulatory A(4) isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldeh yde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, puri fied from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NA D-dependent activities, with a preference for NADP. The structure has been solved to 3.0 Angstrom resolution by molecular replacement. The crystals be long to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point sy mmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in t he cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann f old family of dehydrogenases. However, unlike most dehydrogenases of this f amily, the adenosine T-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossma nn fold and a serine residue located in the S-loop of a symmetry-related mo nomer. While increasing our knowledge of an important photosynthetic enzyme , these results contribute to a general understanding of NADP versus NAD re cognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A(4)-GAPDH is similar to that of the cyto solic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is pecul iar, in that it can actually be considered a dimer of dimers, since monomer s are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from anim als, bacteria, or plants other than spinach. (C) 2001 Academic Press.