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.
Ammendola
S
S
Dipartimento di Biotecnologia, Menarini Ricerche Sud, Pomezia, Italy.
Raucci
G
G
Incani
O
O
Mele
A
A
Tramontano
A
A
Wallace
A
A
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017354:Point Mutation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
D011994:Recombinant Proteins; D018698:Glutamic Acid; D000426:Alcohol Dehydrogenase; D015032:Zinc; D003545:Cysteine