NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69–Ala, Asn-119–His, Ile-175–Ala, Gln-197–Leu, Arg-258–Ala, Gln-287–Glu and His-311–Gln) and double amino acid substitutions (Asn-119–His/His-311–Gln, Gln-287–Glu/His-311–Gln and Gln-287–Glu/Pro-288–Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311–Gln and Gln-287–Glu/His-311–Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate.
The FDH nucleotide sequence has been submitted to the EMBL Nucleotide Sequence Database under the accession no. AJ011046.
Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis
