Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 27, Pages 10946-10951Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1306073110
Keywords
branched-chain amino acid pathway; cofactor imbalance
Categories
Funding
- Gordon and Betty Moore Foundation [GBMF2809]
- Caltech Programmable Molecular Technology Initiative
- German National Academic Foundation
- Kirschstein National Research Service Award [F32GM101792]
- L. Kirschstein National Institutes of Health postdoctoral fellowship [F32GM087102]
- Gordon and Betty Moore Foundation
- Beckman Institute
- Sanofi-Aventis Bioengineering Research Program at Caltech
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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 enzymeswith NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.
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