4KQX

Mutant Slackia exigua KARI DDV in complex with NAD and an inhibitor

Summary for 4KQX

• Entry DOI: 10.2210/pdb4kqx/pdb
• Related: 4KQW
• Descriptor: Ketol-acid reductoisomerase, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, N-HYDROXY-N-ISOPROPYLOXAMIC ACID, ... (6 entities in total)
• Functional Keywords: cofactor switch, rossmann fold, ketol-acid reductoisomerase, acetohydroxyacid isomeroreductase, oxidoreductase-oxidoreductase inhibitor complex, oxidoreductase/oxidoreductase inhibitor
• Biological source: Slackia exigua
• Total number of polymer chains: 2
• Total formula weight: 79883.93

Authors

Brinkmann-Chen, S.,Flock, T.,Cahn, J.K.B.,Snow, C.D.,Brustad, E.M.,Mcintosh, J.A.,Meinhold, P.,Zhang, L.,Arnold, F.H. (deposition date: 2013-05-15, release date: 2013-06-26, Last modification date: 2023-09-20)

Primary citation

Brinkmann-Chen, S.,Flock, T.,Cahn, J.K.,Snow, C.D.,Brustad, E.M.,McIntosh, J.A.,Meinhold, P.,Zhang, L.,Arnold, F.H.
General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.
Proc.Natl.Acad.Sci.USA, 110:10946-10951, 2013

PubMed Abstract: 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.

PubMed: 23776225
DOI: 10.1073/pnas.1306073110

Experimental method

X-RAY DIFFRACTION (1.8 Å)