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. High-resolution structures of a wild-type/variant pair reveal
the molecular basis of the cofactor switch
