Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface.
Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188' and 188-155') across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90 degrees C, in contrast to the wild-type enzyme which precipitates at 52 degrees C. Furthermore, the mutant enzyme has a temperature optimum of 55 degrees C and possesses appreciable enzymatic activity at 65 degrees C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes.
Gokhale
R S
RS
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Agarwalla
S
S
Francis
V S
VS
Santi
D V
DV
Balaram
P
P
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003196:Computer Graphics; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism; D004795:Enzyme Stability; D005971:Glutamates; D018698:Glutamic Acid; D006358:Hot Temperature; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D013912:Threonine; D013940:Thymidylate Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D005971:Glutamates; D046911:Macromolecular Substances; D013912:Threonine; D018698:Glutamic Acid; D013940:Thymidylate Synthase