Genetically engineered proteins with two active sites

Nowadays the technology is enjoying such a high level of sophistication that it has enabled the generation of a spectacular diversity of programmable catalysts with finely orchestrated activities.
Published in Chemistry
Genetically engineered proteins with two active sites

We have explored how meticulous chemical and biochemical design, supported by computational and structural methods, can allow producing better catalysts, which combine biological and new-to-nature chemical transformations skills. This combination thereof may represent a valuable alternative to expand the joined use of enzymes and catalytic metals, particularly for cascade reactions.

In a first step, inspired by recent advances in active site design in protein scaffolds [1] and in inorganic catalysis [2], we imagine enzyme designs where multiple active sites, natural and artificial, work synergistically to yield a proficient biocatalyst. This approach is somehow challenging Nature’s evolution. Indeed, even if Nature has endowed enzymes with the ability to perform chemical reactions with an exquisite proficiency, natural evolution has restricted to one active site per enzyme.

From our earlier work, we know how to computationally prepare an enzyme with two biological active sites, one natural and one artificial [3]. Now, in a work published in Nature Catalysis [4], we further demonstrate that a real synergy between a natural and an artificially-introduced site with an appropriated configuration can significantly promote catalytic turnover (up to 5000-fold), enantio-selectivity and substrate scope of an enzyme. Counter-intuitively, the optimal thermal range gets also expanded by more than 20ºC when a second biological site is added! Proteins with two active sites of biological origin for improved natural bio-catalysis can be from now on designed. We named this design as PluriZymes (the Latin root pluri: multiplicity).

In a second step, we were convinced that it must be possible to find ways around a PluriZyme design to replace one of the biological active sites by an abiological one, so that to design a protein with biological and abiological sites capable of working in concert. We found the right strategy upon reading the seminal work of Wilson and Whitesides [5] and the recent investigations by Ward [6] and Roelfes [7], which show that catalytic metal species can be introduced in a protein scaffold.

We decided to approach the bioconjugation of the PluriZyme system by exploiting the inclination of the enzyme to react with specially designed inhibitors [8]. This reaction, however, does not lead to activity death but to the introduction of a new chemocatalytic center via the presence, on the inhibitor, of a catalytically competent metal ion. Through this methodology, we have developed enzyme scaffolds with two active sites, with bio- and chemo-catalytic activities, respectively. We demonstrate that those artificial catalysts display versatile catalytic activities and perform reactions not supported by the original enzyme as well as new-to-nature reactions.

The exciting implication of our discovery is that adding multiple biological active sites will bring the possibility to improve or add new properties to enzyme scaffolds, while at the same time serving as starting point for a new generation of catalysts with dual bio- and chemo-catalytic properties. The big question is: how important is the methodology herein described in catalysis? The first thing we can say is that PluriZymes have been a surprise to others in the enzymology community, who didn’t expect to see such a development. It also raises questions about how important adding new biological and abiological sites working in synergy are in determining new reactivity. Further computational, structural and experimental studies will help us to determine the answers.

Written by Manuel Ferrer, Patrick Shahgaldian, Víctor Guallar and Julia Sanz.

[1]    Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190-195 (2008)
[2]    Shamzhy, M., Opanasenko, M., Concepción, P. & Martínez, A. New trends in tailoring active sites in zeolite-based catalysts. Chem Soc Rev 48, 1095-1149 (2019).
[3]    Santiago, G. et al. Rational engineering of multiple active sites in an ester hydrolase. Biochemistry 57, 2245-2255 (2018).
[4]    Sandra, A. et al. Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis. Nature Catalysis, in press.
[5]    Wilson, M.E. & Whitesides, G.M. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(I) moiety. J Am Chem Soc 100, 306-307 (1978).
[6]    Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661-665 (2013).
[7]    Bos, J. et al. Enantioselective artificial metalloenzymes by creation of a novel active site at the protein dimer interface. Angew Chem Int Ed Engl 51, 7472-7475 (2012).
[8]    Zollner H. Handbook of enzyme inhibitors (Wiley‐VCH Verlag GmbH), (1999).

Legend to figure: The figure exemplifies how a natural enzyme (left) can be transformed into a Plurizyme through biochemical design (center) and later through chemical design and bioconjugation into a catalyst which combine biological and new-to-nature chemical transformations (right). The figure has been made with Pymol.

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