Brinkmann-Chen, Sabine;
Flock, Tilman;
Cahn, Jackson K. B.;
Snow, Christopher D.;
Brustad, Eric M.;
Mclntosha, John A.;
Meinhold, Peter;
Zhang, Liang;
Arnold, Frances H.
General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH
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Media type:
E-Article
Title:
General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH
Contributor:
Brinkmann-Chen, Sabine;
Flock, Tilman;
Cahn, Jackson K. B.;
Snow, Christopher D.;
Brustad, Eric M.;
Mclntosha, John A.;
Meinhold, Peter;
Zhang, Liang;
Arnold, Frances H.
Published:
National Academy of Sciences, 2013
Published in:
Proceedings of the National Academy of Sciences of the United States of America, 110 (2013) 27, Seite 10946-10951
Language:
English
ISSN:
0027-8424
Origination:
Footnote:
Description:
<p>To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a caseby-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.</p>