Effects of disulphide bridges on the activity and stability of the formate dehydrogenase from <Emphasis Type="Italic">Candida methylica</Emphasis>

Wild-type cmFDH contains no cystines, hence it is a good candidate to test the hypothesis that thermostability can be achieved by introducing new disulphide bridges. Three cysteine double mutants of cmFDH were designed, using a homology model reported previously, to introduce cystine bridges in the C-domain (T169C–T226C) in the N-domain (V88C–V112C) and between the two monomers (M156C–L159C) to form two cystine bridges across the dimer interface. These mutants were constructed and the proteins were over-expressed in E. coli. The mutants V88C–V112C and M156C–L159C lost FDH activity. The mutant T169C–T226C was both less active and less thermostable than wild-type FDH.