title: Complete reversal of coenzyme specificity of isocitrate dehydrogenase from Haloferax volcanii.
abstract: Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP+ to NAD+ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.
authors.affiliation: División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain.
authors.forename: Adoración
authors.initials: A
authors.lastname: Rodríguez-Arnedo
authors.forename: Mónica
authors.initials: M
authors.lastname: Camacho
authors.forename: Francisco
authors.initials: F
authors.lastname: Llorca
authors.forename: María-José
authors.initials: MJ
authors.lastname: Bonete
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D003067:Coenzymes / Q000737:chemistry*; D019622:Haloferax volcanii / Q000201:enzymology* / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000493:pharmacokinetics; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D019843:Archaeal Proteins; D003067:Coenzymes; D050505:Mutant Proteins; D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase