3-DIMENSIONAL STRUCTURE PREDICTION OF THE NAD BINDING-SITE OF PROTON-PUMPING TRANSHYDROGENASE FROM ESCHERICHIA-COLI

A three-dimensional structure of the NAD site of Escerichia coli trans hydrogenase has been predicted. The model is based on analysis of cons erved residues among the transhydrogenases from five different sources , homologies with enzymes using NAD as cofactors or substrates, hydrop hilicity profiles, and secondary structure predictions. The present mo del supports the hypothesis that there is one binding site, located re latively close to the N-terminus of the alpha-subunit. The proposed st ructure spans residues alpha 145 to alpha 287, and it includes five be ta-strands and five alpha-helices oriented in a typical open twisted a lpha/beta conformation. The amino acid sequence following the GXGXXG d inucleotide binding consensus sequence (residues alpha 172 to alpha 17 7) correlates exactly to a typical fingerprint region for ADP binding beta alpha beta folds in dinucleotide binding enzymes. In the model, a spartic acid (alpha 195 forms hydrogen bonds to one or both hydroxyl g roups on the adenosine ribose sugar moiety. Threonine alpha 196 and al anine alpha 256, located at the end of beta B and beta D, respectively , create a hydrophobic sandwich with the adenine part of NAD buried in side. The nicotinamide part is located in a hydrophobic cleft between alpha A and beta E. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support th e predicted structure, and no residue crucial for proton pumping was d etected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great va lue for further experimental design in trying to elucidate the mechani sm of the energy-linked proton pump. (C) 1995 Wiley-Liss,Inc.