"Quasi flexible" automatic docking processing for studying stereoselective recognition mechanisms, part 2: Prediction of DeltaDeltaG of complexation and 1H-NMR NOE correlation.

The purpose of this work is to apply the global molecular interaction evaluation ("Glob-MolInE") computational protocol to the study of two molecular complexes characterized by a chiral selector and a couple of enantiomeric selectands experimentally known to give large difference in the free energy of complexation much higher than the experimental error normally associated to the molecular mechanic calculations. We have considered the well known diastereomeric complexes between the selector (S)-N-(3,5-dinitrobenzoyl)-leucine-n-propylamide (S)-1 and the selectands (R) or (S)-N-(2-naphthyl)-alanine methyl ester 2, widely studied by enantioselective HPLC, NMR and X-ray. The experimental difference of free energy of complexation between [(S)-1*(R)-2] and [(S)-1*(S)-2] (-1.34 kcal/mol) was reproduced by the new computational protocol with an excellent confidence error. Detailed results about the conformational search, the "quasi-flexible" docking and the thermodynamic estimation are presented in this work. A remarkable correlation between the theoretical results and experimental data (NOE measurements, X-ray crystallographic structure of the [(S)-1*(S)-2] complex and the free energy of complexation) supports the validity of the computational approach and underline the importance of the conformational multiplicity in the definition of the macroscopic properties of the complex in solution.

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability.

The alcohol dehydrogenase gene from the thermophilic archaeum Sulfolobus solfataricus has been subcloned and expressed in Escherichia coli under the control of the T7 inducible promoter. The recombinant protein shows properties analogous to those of the native enzyme, including thermostability, despite the fact that E.coli does not post-translationally modify two lysine residues which are N-epsilon-methylated in the native enzyme. We constructed a 3-D model of the S.solfataricus alcohol dehydrogenase using the known structure of its isozyme from horse liver as a template. Our analysis of the structural zinc binding site suggested that this site is present and functional in the S.solfataricus enzyme and that a glutamate ligand can contribute to thermostability by influencing electrostatic interactions around the metal centre. To investigate this hypothesis, we constructed, expressed and characterized a mutant where the glutamate is replaced by a cysteine, thus restoring the zinc binding site of mesophilic alcohol dehydrogenases. The mutant shows the same activity but a reduced thermostability with respect to the wild-type recombinant protein, as suggested by our model.