Active-site characterization of Candida boidinii formate dehydrogenase.
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.
Labrou
N E
NE
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece. lambrou@aua.gr
Rigden
D J
DJ
D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism
D005561:Formates; D009243:NAD; C030544:formic acid; D005560:Formate Dehydrogenases; C020644:Formate dehydrogenase (cytochrome)