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

Brinkmann-Chen, Sabine and Flock, Tilman and Cahn, Jackson K. B. and Snow, Christopher D. and Brustad, Eric M. and McIntosh, John A. and Meinhold, Peter and Zhang, Liang and Arnold, Frances H. (2013) General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proceedings of the National Academy of Sciences of the United States of America, 110 (27). pp. 10946-10951. ISSN 0027-8424. PMCID PMC3704004. doi:10.1073/pnas.1306073110.

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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 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.

Item Type:Article
Related URLs:
URLURL TypeDescription DOIArticle CentralArticle
Brinkmann-Chen, Sabine0000-0002-5419-4192
Snow, Christopher D.0000-0002-7690-3519
Brustad, Eric M.0000-0002-7031-5433
McIntosh, John A.0000-0002-9487-490X
Zhang, Liang0000-0003-3115-1752
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2013 National Academy of Sciences. Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved May 21, 2013 (received for review March 29, 2013). Published online before print June 17, 2013. We thank Dr. Jens Kaiser and Dr. Pavle Nikolovski for their continued support. This publication was supported by the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative, the German National Academic Foundation (to T.F.), a Ruth L. Kirschstein National Research Service Award (F32GM101792) (to J.A.M.), and a Ruth L. Kirschstein National Institutes of Health postdoctoral fellowship (F32GM087102) (to E.M.B.). The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech. Author contributions: S.B.-C., T.F., J.K.B.C., C.D.S., P.M., and F.H.A. designed research; S.B.-C., T.F., J.K.B.C., and L.Z. performed research; J.A.M. contributed new reagents/analytic tools; S.B.-C., T.F., J.K.B.C., C.D.S., E.M.B., and F.H.A. analyzed data; S.B.-C., T.F., J.K.B.C., C.D.S., E.M.B., and F.H.A. wrote the paper. Conflict of interest statement: F.H.A. is cofounder of Gevo, Inc. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors for wild-type Slackia exigua Se_KARI and mutant S. exigua Se_KARIDDV have been deposited in the Protein Data Bank (PDB), (PDB ID codes 4KQW and 4KQX, respectively).
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
Caltech Programmable Molecular Technology InitiativeUNSPECIFIED
Deutscher Akademischer Austauschdienst (DAAD)UNSPECIFIED
NIH Predoctoral FellowshipF32GM101792
NIH Predocotral FellowshipF32GM087102
Caltech Beckman InstituteUNSPECIFIED
Caltech Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Subject Keywords:branched-chain amino acid pathway; cofactor imbalance
Issue or Number:27
PubMed Central ID:PMC3704004
Record Number:CaltechAUTHORS:20130905-104306564
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41113
Deposited By: Tony Diaz
Deposited On:17 Sep 2013 20:38
Last Modified:10 Nov 2021 04:26

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