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Publication Abstract from PubMed

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.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.,Brinkmann-Chen S, Flock T, Cahn JK, Snow CD, Brustad EM, McIntosh JA, Meinhold P, Zhang L, Arnold FH Proc Natl Acad Sci U S A. 2013 Jul 2;110(27):10946-51. doi:, 10.1073/pnas.1306073110. Epub 2013 Jun 17. PMID:23776225^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.