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Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from Haloferax volcanii

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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.

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Abbreviations

IC:

isocitrate

ICDH:

isocitrate dehydrogenase

IMDH:

isopropylmalate dehydrogenase

LB:

Luria-Bertani

NAD+ :

β-nicotinamide adenine dinucleotide phosphate

NADP+ :

β-nicotinamide adenine dinucleotide phosphate

NADPH:

reduced β-nicotinamide adenine dinucleotide phosphate

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Authors and Affiliations

  1. División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080, Alicante, Spain

    Adoración Rodríguez-Arnedo, Mónica Camacho, Francisco Llorca & María-José Bonete

Authors
  1. Adoración Rodríguez-Arnedo
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  2. Mónica Camacho
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  3. Francisco Llorca
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  4. María-José Bonete
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Correspondence to María-José Bonete.

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Rodríguez-Arnedo, A., Camacho, M., Llorca, F. et al. Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from Haloferax volcanii . Protein J 24, 259–266 (2005). https://doi.org/10.1007/s10930-005-6746-8

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  • DOI: https://doi.org/10.1007/s10930-005-6746-8

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