Effects of disulphide bridges on the activity and stability of the formate dehydrogenase from Candida methylica.
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.
Karagüler
Nevin Gül
NG
Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey. karaguler@itu.edu.tr
Sessions
Richard B
RB
Clarke
Anthony R
AR
D001665:Binding Sites; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*
D004220:Disulfides; D005560:Formate Dehydrogenases