Uncovering rare NADH-preferring ketol-acid reductoisomerases

All members of the ketol-acid reductoisomerase (KARI) enzyme family characterized to date have been shown to prefer the nicotinamide adenine dinucleotide phosphate hydride (NADPH) cofactor to nicotinamide adenine dinucleotide hydride (NADH). However, KARIs with the reversed cofactor preference are d...

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Bibliographic Details
Published in:Metabolic engineering 2014-11, Vol.26, p.17-22
Main Authors: Brinkmann-Chen, S., Cahn, J.K.B., Arnold, F.H.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Cofactor specificity
Enzyme Activation
Ketol acid reductoisomerase
Ketol-Acid Reductoisomerase - chemistry
Molecular Sequence Data
NAD - chemistry
NADH
NADPH
Protein Binding
Sequence Alignment - methods
Sequence Analysis, Protein - methods
Substrate Specificity
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Summary:All members of the ketol-acid reductoisomerase (KARI) enzyme family characterized to date have been shown to prefer the nicotinamide adenine dinucleotide phosphate hydride (NADPH) cofactor to nicotinamide adenine dinucleotide hydride (NADH). However, KARIs with the reversed cofactor preference are desirable for industrial applications, including anaerobic fermentation to produce branched-chain amino acids. By applying insights gained from structural and engineering studies of this enzyme family to a comprehensive multiple sequence alignment of KARIs, we identified putative NADH-utilizing KARIs and characterized eight whose catalytic efficiencies using NADH were equal to or greater than NADPH. These are the first naturally NADH-preferring KARIs reported and demonstrate that this property has evolved independently multiple times, using strategies unlike those used previously in the laboratory to engineer a KARI cofactor switch. •Available KARI sequences searched for those predicted to effectively utilize NADH.•Eight novel KARIs characterized: four are bi-specific (use NADH and NADPH) and four prefer NADH.•These are the first natural NADH-utilizing KARIs reported.•Nature found different cofactor re-engineering solutions.
ISSN:1096-7176
1096-7184
DOI:10.1016/j.ymben.2014.08.003