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Protein Engineering vol. 8 no. 1 pp. 31-37, 1995
© 1995 Oxford University Press

RESEARCH-ARTICLE

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

Sergio Ammendola1, Giuseppe Raucci, Ottaviano Incani2, Antonio Mele, Anna Tramontano3 and Andrew Wallace3

Dipaitimento di Biotecnologia, Menarini Ricerche Sud Via Tito Speri, 10-00040 Pomezia 2Laboratorio di Chimica Computazionale Tecnofarmaci S.c.p.A., Plazza Indipendenza, 25-00040 Pomezia 3Istituto di Ricerche di Biologia Molecolare (IRBM) P.Angeletti Via Pontina Km, 30.600-00040 Pomezia, Italy

1To whom correspondence should be addressed

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-{varepsilon}-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 andfunctional in the S.solfataricus enzyme and that a glutamate ligand can contribute to thermostability by influencing electrostatic interactions around the metal centre. To investigate thishypothesis, 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. Themutant shows the same activity but a reduced thermostability with respect to the wild-type recombinant protein, as suggested by our model.

Keywords: alcohol dehydrogenase/metal binding site/modelling/rational mutagenesis/thermostability

Received May 23, 1994; revised September 12, 1994; accepted September 23, 1994.


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