General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH
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.
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), www.pdb.org (PDB ID codes 4KQW and 4KQX, respectively).Attached Files
Published - PNAS-2013-Brinkmann-Chen-10946-51.pdf
Supplemental Material - pnas.201306073SI.pdf
Files
| Name | Size | Download all |
|---|---|---|
|
md5:f673210324bbafe963170280688020b4
|
61.2 kB | Preview Download |
|
md5:5f9fd4ae0d0f100a499af71721174a65
|
1.5 MB | Preview Download |
Additional details
- PMCID
- PMC3704004
- Eprint ID
- 41113
- Resolver ID
- CaltechAUTHORS:20130905-104306564
- GBMF2809
- Gordon and Betty Moore Foundation
- Caltech Programmable Molecular Technology Initiative
- Deutscher Akademischer Austauschdienst (DAAD)
- F32GM101792
- NIH Predoctoral Fellowship
- F32GM087102
- NIH Predocotral Fellowship
- Caltech Beckman Institute
- Caltech Sanofi-Aventis Bioengineering Research Program
- Created
-
2013-09-17Created from EPrint's datestamp field
- Updated
-
2021-11-10Created from EPrint's last_modified field