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 archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.