A map of the rubisco biochemical landscape
- Creators
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Prywes, Noam
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Philips, Naiya R.
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Oltrogge, Luke M.
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Lindner, Sebastian
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Tsai, Yi-Chin Candace
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de Pins, Benoit
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Cowan, Aidan E.
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Taylor-Kearney, Leah J.
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Chang, Hana A.
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Hall, Laina N.
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Bellieny-Rabelo, Daniel
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Nisonoff, Hunter M.
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Weissman, Rachel F.
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Flamholz, Avi I.
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Ding, David
- Bhatt, Abhishek Y.
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Shih, Patrick M.
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Mueller-Cajar, Oliver
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Milo, Ron
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Savage, David F.
Abstract
Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.
Copyright and License
Acknowledgement
We thank Niv Antonovsky and Arren Bar-Even for taking part in formulating the basis for this work as well as Naama Tepper and Shira Amram for originally conceiving of and producing the Δrpi strain respectively. We thank Philip Romero, Nat Thompson, Leon Fedotov, Orren Saltzman, Eden Prywes, Stacia Wyman, Bin Yu and Jack Desmarais for essential help in the process of data analysis. For their assistance in the process of generating and validating the DMS library we thank Andrew Glazer, Kenneth Matreyek, Jesse Bloom and Kim Reynolds. Additionally we thank Julia Tartaglia for the use of her sequencing primers and Netra Krishnappa for assistance in running NGS samples. We would like to thank Elaine Meng for assistance using ChimeraX. Finally we thank Flora Wang for technical assistance over the weekends.
Funding
National Institutes of Health grant K99GM141455–01 (NP)
DFS is an Investigator of the Howard Hughes Medical Institute
U. S. Department of Energy, Physical Biosciences Program, Award Number DE-SC0016240 (DFS)
Data Availability
All data are available in the main text or the supplementary materials.
Contributions
Conceptualization: NP, AIF, DFS
Methodology: NP, NRP, LMO, SL, DD, OMC, RM, DFS
Investigation: NP, NRP, SL, YCT, BdP, AEC, LJTK, HAC, LNH, DBR, HMN, RFW, AYB
Visualization: NP, LMO, SL, DFS
Funding acquisition: NP, DFS
Project administration: NP, DFS
Supervision: NP, PMS, OMC, RM, DFS
Writing – original draft: NP, LNH, AIF, DFS
Conflict of Interest
DFS is a co-founder and scientific advisory board member of Scribe Therapeutics.
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Additional details
- PMCID
- PMC11030240
- National Institutes of Health
- K99GM141455–01
- Howard Hughes Medical Institute
- United States Department of Energy
- DE-SC0016240