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Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis


Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on proteins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel–Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (≥83%; e.e. >99.9%) were achieved in one pot with this artificial multifunctional metalloenzyme.

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Fig. 1: General concept for engineering proteins with two active sites.
Fig. 2: Biocatalytic advantages of having two biological active sites.
Fig. 3: Crystal structure of EH1AB1.
Fig. 4: Absorption spectra of modified and unmodified sub-enzymes.
Fig. 5: Electrochemical characterization of the chemo-biocatalysts.
Fig. 6: Image representing the chemo-biocatalyst EH1AB1C-B generated in this study and its utilization in two model reactions.
Fig. 7: One-pot synthesis of 1,4-naphthoquinone from 1-napthyl acetate catalysed by EH1AB1C-B.
Fig. 8: One-pot synthesis of 3-phenylbutyric acid from vinyl crotonate and benzene catalysed by EH1AB1C-B.

Data availability

The atomic coordinates have been deposited in the Protein Data Bank under accession numbers 6I8F, 6RB0 and 6RKY. All other data are available from the authors upon reasonable request.


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This work was funded by grant ‘INMARE’ from the European Union’s Horizon 2020 (grant agreement no. 634486), grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), CTQ2016-79138-R, BIO2016-76601-C3-1-R, BIO2016-76601-C3-3-R, BIO2017-85522-R, RTI2018-095166-B-I00 and RTI2018-095090-B-100 from the Ministerio de Economía y Competitividad, the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI), the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). P.N.G. and R.B. acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant No. BB/M029085/1) and the Centre of Environmental Biotechnology Project and the Supercomputing Wales project, which are partly funded by the European Regional Development Fund (ERDF) through the Welsh Government. The authors gratefully acknowledge the financial support provided by the ERDF. C.C. thanks the Ministerio de Economía y Competitividad and FEDER for a Ph.D. fellowship (Grant BES-2015-073829). J.L.G.-A. thanks the support of the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). I.C.-R. thanks the Regional Government of Madrid for a fellowship (PEJ_BIO_AI_1201). The authors would like to acknowledge S. Ciordia and M. C. Mena for MALDI-TOF/TOF analysis. We thank the staff of both the European Synchrotron Radiation Facility (ESRF, Grenoble, France), for providing access and technical assistance at beamline ID30A-1/MASSIf-1, and the Synchrotron Radiation Source at Alba (Barcelona, Spain), for assistance at BL13-XALOC beamline. The authors would also like to acknowledge M. J. Vicente and M. A. Pascual at the Servicio Interdepartamental de Investigación (SIDI) of the Autonomous University of Madrid for the ESI-MS analyses.

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Authors and Affiliations



S.A., G.S. and I.C.-R. contributed equally to this work. The manuscript was written through contributions from M.F., V.G., J.S.-A. and P.S. All the authors have given approval to the final version of the manuscript. S.A., C.C., L.F.-L., M.M.-M., H.M. and P.N.G. contributed to site-directed mutagenesis and protein expression, purification and characterization. J.M. and A.K.R. coordinated, in collaboration with M.F., the synthesis of the suicide inhibitor. R.B. contributed to the biochemical data analysis. D.R. and C.B. contribute to the GC analyses for enantioselectivity determination. J.L.G.-A. and F.J.P. performed HPLC analysis of the reaction products. G.S. and V.G. conducted the PELE simulations and molecular dynamics. M.P. contributed together with S.A. and L.F.-L. to the electrochemical measurements and discussion. I.C.-R. and J.S.-A. performed the crystallization and X-ray structure determinations. M.B. contributed to the development of the protocol for the inhibition procedure. M.F. and V.G. conceived the plurizyme work, and M.F. and P.S. conceived the metamorphosis of the enzymatic to the chemical catalyst. M.F. wrote the initial draft of the manuscript, which was further supplemented through contributions from V.G., J.S.-A. and P.S.

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Correspondence to Julia Sanz-Aparicio, Víctor Guallar or Manuel Ferrer.

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Supplementary Information

Supplementary Notes 1–12, Figures 1–36, Tables 1–8, methods and references.

Reporting Summary


Atomic coordinates for PDB ID 6I8F


Atomic coordinates for PDB ID 6RB0


Atomic coordinates for PDB ID 6RKY

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Alonso, S., Santiago, G., Cea-Rama, I. et al. Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis. Nat Catal 3, 319–328 (2020).

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