Active-site characterization of Candida boidinii formate dehydrogenase

NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was clon ed and expressed to a high level in Escherichia coli (20% df soluble E. col i protein). Molecular modelling studies were used to create a three-dimensi onal model of C. boidinii FDH, based on a known structure of the Pseudomona s sp. 101 enzyme. This model was used for investigating the catalytic mecha nism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were cha racterized by steady-state kinetic analysis: the wild type as well as 10 mu tants involving single (Phe-69-Ala, Asn-119-His, Ile-175-Ala, Gln-197-Leu, Arg-258-Ala, Gln-287-Glu and His-311-Gin) and double amino acid substitutio ns (Asn-119-His/His311-Gln, Gln-287-Glu/His311-Gln and Gln-287-Glu/Pro-288- Thr). The kinetic results of the mutant enzymes provide the first experimen tal support that hydrophobic patches, formed by Phe-69 and Ile-175, destabi lize substrates and stabilize products. Also, the key role of Arg-258 in st abilization 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-G lu/His-311-Gln mutants, combined with molecular modelling, suggest that ste ric 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 th e proper orientation for productive binding of formate.