Stabilization of NAD-dependent formate dehydrogenase from Candida boidiniiby site-directed mutagenesis of cysteine residues

The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Ca ndida boidinii was cloned by PCR using genomic DNA as a template. Expressio n of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inac tivation process, both cysteine residues in the primary structure of the en zyme have been exchanged by site-directed mutagenesis using a homology mode l based on the 3D structure of FDH from Pseudomonas sp. 101 and from relate d dehydrogenases. Compared to the wt enzyme, most of the mutants were signi ficantly more stable towards oxidative stress in the presence of Cu(II) ion s, whereas the temperature optima and kinetic constants of the enzymatic re action are not significantly altered by the mutations. Determination of the T-m values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (T-m 57 degrees C), whereas the T-m values of the mutant enzymes vary in the range 44-52 degre es C. Best results in initial tests concerning the application of the enzym e for regeneration of NADH in biotransformation of trimethyl pyruvate to lt ert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, whic h are significantly more stable than the wt enzyme.