Department of Inorganic Chemistry and the Laboratory of Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
D-Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12, GAP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate to form 1,3-diphosphoglycerate in the presence of NAD+ and inorganic phosphate. GAP-DH thus generates two molecules of NADH per molecule of glucose during glycolytic degradation. The Sacchromyces cerevisia (Baker's yeast) version of the enzyme (yGAP-DH) has characteristics similar to those reported for GAP-DH from other sources. All known NAD+-dependent Glyceraldehyde-3- phosphate Dehydrogenases are composed of four identical subunits, have molecular weights of around 145,000 and bind four moles of the NAD+ cofactor. The three dimensional crystal structure of holo-yGAP-DH from has been now determined to 2.1 Å resolution. The solution of the structure was achieved by molecular replacement using the structure of holo-GAP-DH from Bacillus stearothermophilus as a reference model. The structure was refined to an R- factor of 16.3% for 56,193 reflections in the range of 30.0 - 2.1 Å resolution. The asymmetric unit in the crystal contains two monomers of GAP-DH which are related by an approximate 2-fold symmetry axis, and the GAP-DH tetramer is further constructed from such dimers by the unit cell crystallographic symmetry. The yGAP-DH monomer is built of two domains, the NAD+ domain and the catalytic domain. Similar to the other known GAP-DH's the NAD+ domain is constructed of parallel beta-sheet strands mixed with alpha-helices in a "Rossmann Fold". The catalytic domain is constructed of 8 beta-sheet strands, 4 of which form the anti-parallel sheet of the catalytic core, and an extra loop (the "S-loop") which plays an important role in the binding of NAD+. The overall structure of yGAP-DH is quite similar to the structure of the Bacillus GAP-DH with a root-mean-square (rms) deviation of 0.74 Å for the Ca positions between the NAD+ domains of the two structures and even smaller rms deviation between the catalytic domains of the two structures. Comparison of the structures of the mesophilic yGAP-DH to those of the thermophilic GAP-DH from B. stearothermophilus and the hyperthermophilic GAP-DH from Thermotoga maritima indicate that specific intramolecular ion pairs in these tetramers are the main factor for the differences in the observed thermostability.