Uncovering rare NADH-preferring ketol-acid reductoisomerases

S Brinkmann-Chen; J K B Cahn; F H Arnold

doi:10.1016/j.ymben.2014.08.003

Uncovering rare NADH-preferring ketol-acid reductoisomerases

Metab Eng. 2014 Nov:26:17-22. doi: 10.1016/j.ymben.2014.08.003. Epub 2014 Aug 27.

Authors

S Brinkmann-Chen¹, J K B Cahn², F H Arnold³

Affiliations

¹ California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: sabine@cheme.caltech.edu.
² California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: jcahn@caltech.edu.
³ California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA. Electronic address: frances@cheme.caltech.edu.

PMID: 25172159
DOI: 10.1016/j.ymben.2014.08.003

Abstract

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.

Keywords: Cofactor specificity; Ketol acid reductoisomerase; NADH; NADPH.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Binding Sites
Enzyme Activation
Ketol-Acid Reductoisomerase / chemistry*
Molecular Sequence Data
NAD / chemistry*
Protein Binding
Sequence Alignment / methods*
Sequence Analysis, Protein / methods*
Substrate Specificity

Substances

NAD
Ketol-Acid Reductoisomerase