news & views

ARTIFICIAL Synergistic in an artifcial The introduction of single abiological catalytic groups enables enzymes to catalyse new-to-nature chemical transformations. Now, this concept is extended to two abiological groups in a single protein scafold to allow synergistic catalysis in a stereoselective Michael addition reaction. Xinkun Ren and Rudi Fasan

iocatalysis constitutes an attractive transformation can be tuned by protein development of artificial enzymes, in which and competitive technology for engineering1,2. However, natural enzymes an abiological catalytic group, often in the Bthe synthesis of chiral molecules, catalyse a much narrower spectrum form of a synthetic organometallic complex, pharmaceuticals, and other high-value of chemical reactions compared to is covalently or non-covalently embedded compounds, as enzymes often display chemocatalytic methods, which limits their into a protein scaffold, thereby equipping high degrees of stereoselectivity, function scope for many synthetic applications. the protein with a new catalytic function3. under ambient conditions, and their Toward overcoming this limitation, a More recently, an alternative strategy has activity and selectivity toward a particular major approach has entailed the design and relied on the redesign and/or engineering of

a F93’

N19 W96’ M8

M89’ M89 pAF15’ pAF15 N19’ W96 M8’

pAF-containing LmrR variant

bc

O O H H N N ......

N N

Cu N NH Ar O 2 NH Cu N O3N NO3 N N p-aminophenylalanine Cu(1,10-phenanthroline)(NO3)2

R

d

O O 1 2 1 2 a: R = Ph, R = H e: R = p-MeOPh, R = CH3 1 LmrR_V15pAF variants R1 1 2 1 2 N R CHO N * b: R = Ph, R = CH3 f: R = p-MeOPh, R = H + R2 1 2 1 2 c: R = Ph, R = nPr g: R = p-ClPh, R = CH3 Cu(II)-phenanthroline N CHO 1 2 1 2 N 2 d: R = Ph, R = Ph h: R = 3-thienyl, R = CH3 R *

Fig. 1 | LmrR-based artificial enzyme for asymmetric Michael addition. a, of p-aminophenylalanine (pAF)-containing LmrR protein scaffold. b,c, Abiological components (b) and putative mechanism (c) of the artificial enzyme. d, scope of LmrR_V15pAF/Cu-phen catalysed Michael addition reaction. Credit: panel c adapted with permission from ref. 5, Springer Nature Ltd.

184 Nature Catalysis | VOL 3 | March 2020 | 184–185 | www.nature.com/natcatal news & views natural to realize abiological To create the artificial enzyme, determined to support about 10–20 4 transformations . Both approaches have the authors started from a previously turnovers with a catalytic efficiency (kcat/KM) produced attractive avenues for creating developed LmrR variant in which of 0.1 M–1 s–1. The latter remains orders of biocatalysts capable of mediating chemical p-aminophenylalanine is introduced in magnitude lower than that of natural or reactions beyond those carried out by close proximity to the predicted cofactor engineered enzymes used in biocatalysis. natural enzymes. binding pocket defined by the central Further optimization via protein engineering A key feature underlying the catalytic tryptophan residues (Fig. 1a). Next, the along with elucidation of the stereochemical proficiency of natural enzymes is their authors tested the relative efficiency of preference of the biocatalyst, which remains ability to exploit multiple catalytic various organometallic copper complexes currently undefined, are expected to provide mechanisms in concert to accelerate the non-covalently bound to the protein, potential avenues for further development rate of a chemical reaction by several orders resulting in the identification of Cu(1,10- and refinement of this artificial enzyme in of magnitude (for example, by 105- to 1020- phenanthroline) as the most effective choice the future. While the LmrR-based enzyme is fold). Previous efforts toward creating for productive generation of the enolate currently assembled in vitro, acquiring the artificial enzymes have focused on the nucleophile. The resulting protein complex capability to assemble this system directly introduction of a single, abiological was found to be able to catalyse a Michael in a cell, as has recently become possible for catalytic group, such as an organometallic addition reaction between acrolein and other types of artificial enzymes6,7, would complex or an organocatalytic group, an imidazolyl-ketone in moderate yield facilitate these protein engineering efforts. into a protein scaffold3. In these systems, (36%) but with promising enantioselectivity Prior to this work, genetically encoded concerted catalysis has been achieved (86%) (Fig. 1c). Importantly, additional non-canonical amino acids have been in some cases by combining the unique experiments demonstrated the synergistic incorporated into engineered and artificial reactivity of the abiological catalytic group action of the two abiological catalytic enzymes to alter their substrate scope, with manipulation of the groups, that is, the pAF residue and the tune their reactivity, or create and residues surrounding the active centre, protein-bound Cu-complex, in promoting append new catalytic centres8. The work by resulting in improved catalytic activity and/or this reaction, along with the superior activity Zhi Zhou and Gerard Roelfes expands upon refined chemo- and stereoselectivity.3 and enantioselectivity of the artificial this concept by demonstrating the value of Now, writing in Nature Catalysis, Zhi Zhou enzyme compared to commonly used achieving synergistic catalysis in an artificial and Gerard Roelfes report an important (chiral) amine-based synthetic catalysts. enzyme through the combination of a non- development in the field of artificial enzyme The substrate scope of this biocatalyst canonical amino acid and an abiological design, in which two abiological catalytic could then be extended to catalyse this organometallic complex. This strategy is groups are designed to act in concert toward transformation in the presence of various expected to prove valuable toward creating catalysing a stereoselective Michael addition α,β-unsaturated aldehydes and 2-acyl and expanding the catalytic repertoire of reaction5 (Fig. 1c). imidazole derivatives as the Michael acceptor artificial enzymes in the context of other The artificial enzyme reported by the and donor substrates, respectively (Fig. 1c). valuable chemical transformations. ❐ authors is based on a bacterial multidrug This reaction generates two new stereocentres resistance regulator protein, called LmrR, and thus four possible products, posing the Xinkun Ren and Rudi Fasan ✉ which forms a homodimeric complex problem of controlling both the diastereo- Te Department of Chemistry, University of featuring a hydrophobic central cavity lined and enantioselectivity of the reaction. In Rochester, Rochester, NY, USA. up by two tryptophan residues (Fig. 1a). most cases, the LmrR-based enzyme was ✉e-mail: [email protected] Building upon previous work, the authors found to yield the desired Michael addition exploited this cavity to non-covalently bind a products with good diastereocontrol (up Published online: 18 March 2020 Cu-complex to the protein, which was meant to 9:1 d.r.) and good-to-excellent levels of https://doi.org/10.1038/s41929-020-0435-z to act as a Lewis acid for the activation of enantioselectivity (up to >99% e.e.), thus References the nucleophilic substrate in the desired demonstrating its functionality across a range 1. Bornscheuer, U. T. et al. Nature 485, 185–194 (2012). Michael addition reaction (Fig. 1b). The of different substrates (Fig. 1c). Through 2. Honig, M., Sondermann, P., Turner, N. J. & Carreira, E. M. Angew. authors further envisioned that synergistic mutagenesis studies, the authors further Chem. Int. Ed. 56, 8942–8973 (2017). catalysis in this reaction could be achieved established that a Met8Val mutation, which 3. Schwizer, F. et al. Chem. Rev. 118, 142–231 (2018). 4. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Curr. Opin by incorporating a non-canonical amino is located near to the Cu-complex binding Biotechnol. 47, 102–111 (2017). acid, p-aminophenylalanine (pAF), in close pocket (Fig. 1a), has a beneficial effect toward 5. Zhou, Z. & Roelfes, G. Nat. Catal. https://doi.org/10.1038/s41929- proximity to the reaction centre. This pAF- increasing the activity and stereoselectivity of 019-0420-6 (2020). 6. Jeschek, M. et al. Nature 537, 661–665 (2016). based abiological catalytic group was meant the enzyme for some of the target reactions. 7. Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. ACS Catal. 7, to enhance the reactivity of the Michael Overall, this study describes an elegant 7629–7633 (2017). acceptor substrate (aldehyde) toward the approach to artificial enzyme design, which 8. Mirts, E. N., Bhagi-Damodaran, A. & Lu, Y. Acc. Chem. Res. 52, Michael addition reaction via iminium could be extended to other systems. A 935–944 (2019). catalysis (Fig. 1b), a mechanism commonly current limitation of the present system Competing interests exploited in organocatalysis. is a modest catalytic activity, as it was The authors declare no competing interests.

Nature Catalysis | VOL 3 | March 2020 | 184–185 | www.nature.com/natcatal 185