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Title: General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

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 case-by-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. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.
 [1] ;  [2] ;  [1] ;  [3] ;  [4] ;  [1] ;  [5] ;  [1] ;  [1]
  1. California Inst. of Technology (CalTech), Pasadena, CA (United States)
  2. California Inst. of Technology (CalTech), Pasadena, CA (United States); MRC Lab. of Molecular Biology, Cambridge (United Kingdom)
  3. California Inst. of Technology (CalTech), Pasadena, CA (United States); Colorado State Univ., Fort Collins, CO (United States)
  4. California Inst. of Technology (CalTech), Pasadena, CA (United States); Univ. of North Carolina, Chapel Hill, NC (United States)
  5. Gevo, Inc., Englewood, CO (United States)
Publication Date:
OSTI Identifier:
Report Number(s):
Journal ID: ISSN 0027-8424
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 110; Journal Issue: 27; Journal ID: ISSN 0027-8424
National Academy of Sciences, Washington, DC (United States)
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; branched-chain amino acid pathway; cofactor imbalance