Complete reversal of coenzyme specificity of isocitrate dehydrogenase from Haloferax volcanii.
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.
Rodríguez-Arnedo
Adoración
A
División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain.
Camacho
Mónica
M
Llorca
Francisco
F
Bonete
María-José
MJ
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
D019843:Archaeal Proteins; D003067:Coenzymes; D050505:Mutant Proteins; D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase