Artificial Metalloenzyme Design with Unnatural Amino Acids and Non

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Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors Yang Yu, Cheng Hu, Lin Xia, and Jiangyun Wang ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03754 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors Yang Yu, † Cheng Hu, ‡ Lin Xia,¶ Jiangyun Wang‡,* AUTHOR ADDRESS †

Tianjin Institute of Industrial Biotechnology,

Chinese Academy of Sciences 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China.

‡

Laboratory of RNA Biology, Institute of Biophysics

Chinese Academy of Sciences 15 Datun Road, Chaoyang District, Beijing, 100101, China.

¶

Center for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China.

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ABSTRACT

There are 20 proteinogenic amino acids and a limited number of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymatic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or non-covalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymatic activities of artificial metalloenzymes.

Key words: Unnatural amino acid, non-native cofactors, genetic code expansion, bioorthogonal reaction, metalloenzyme 1 Introduction Amino acids and cofactors are the basic building blocks of protein-based enzymes. In nature, there are 20 proteinogenic amino acids, which have various side chain structures and functional groups. Amino acid sequence of a protein is encoded by nucleic acids according to the Central dogma.1 Protein synthesis in the cell happens in the protein translation machinery, composed of ribosome, initiation factor, elongation factor, and release factor. The whole process occurs with astonishing speed (10–20 aa/s)2 and fidelity (10-4–10-5 /aa)3. Combinations of these 20


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genetically encoded amino acids, together with approximately 100 cofactors,4 such as metal ions, metallocycles, and flavins, produce the vast structural and functional diversity of proteins. Nature have created potent and diverse catalysts by using natural amino acids and cofactors as basic building blocks through billions of years’ evolution.5-19 Compared to the natural amino acids and cofactors, unnatural amino acids and non-native cofactors provide many more structural and chemical properties. Emerging methods to incorporate unnatural amino acids and non-native cofactors into a protein greatly enhance our ability to expand the activity of natural enzymes and to design novel biocatalysts.7,20-27 Importantly, unnatural amino acids can provide an important “handle” for the precise conjugation of unnatural cofactors through bio-orthogonal reaction--chemical reactions that can occur without interfering with native biochemical processes, providing infinite diversity and exciting new possibilities for enzyme design. 2 Unnatural amino acids for artificial metalloenzyme design 2.1 Genetic code expansion The genetic code expansion method enables site-specific unnatural amino acid incorporation inside the cell (Figure 1).28 An orthogonal tRNA/amino-acyl tRNA synthetase pair is introduced into an organism. The aminoacyl-tRNA synthetase (aaRS) recognizes a specific unnatural amino acid and then acylates the tRNA with the unnatural amino acid. The tRNA recognizes a blank codon (codons that do not encode proteinogenic amino acids), which could be either amber stop codon (UAG), or quadruplet codon, or from genomes with reassigned codons. The tRNA and the aaRS are mutually specific, do not crosstalk with other endogenous tRNA or aaRS, and are compatible with the translation apparatus, forming the orthogonality of the pair. In the translation process, the amino-acylated tRNA enters the ribosome and recognizes the correct


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codon on mRNA with the help of elongation factor Tu (EF-Tu). The unnatural amino acid on the charged tRNA is then transferred to the growing peptide chain in the peptidyl transfer center of the ribosome. Along with other factors in the translational machinery, the specific unnatural amino acid is incorporated into the protein at the position corresponding to the blank codon.

Figure 1. In the native translation process (left), tRNA is charged with the corresponding natural amino acid by aaRS, the anticodon loop of tRNA recognizes corresponding codon on the mRNA, and as a result, the amino acid is added to the peptide chain during translation. Genetic codon expansion (right) introduces orthogonal tRNA and aaRS, which charges the tRNA with a specific unnatural amino acid. tRNA recognizes a reassigned codon (UAG here), and the unnatural amino acid it carries is added to the peptide. Reprinted from ref 23 with permission from Springer.

As a prerequisite for incorporation, the orthogonal tRNA/aaRS pair must be evolved for each unnatural amino acid. Research in the past 15 years has generated more than 180 such pairs, for approximately 200 unnatural amino acids. Liu and Schultz summarized unnatural amino


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acids that can be incorporated through the genetic codon expansion method in 2010.20 A recent review by Davis and coworkers listed ~170 unnatural amino acids, their expression systems and potential applications.29 These amino acids, including the ones reviewed in this article (Figure 2), compose an expanded alphabet for protein design and study, including bio-orthogonal reactions, spectroscopic probes (fluorescent spectroscopy, infra-red spectroscopy, nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, X-ray), posttranslation modifications and protein designs. Here, we review recent progress using unnatural amino acids to design bio-catalysts and to engineer and study enzymes.

Figure 2. Unnatural amino acids used in artificial metalloenzyme design.

2.2 Unnatural amino acid for active site mimicking


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Post-translational modifications (PTM) occur on amino acid residues during or after protein synthesis. It is difficult to control the degree and site of PTM in the as-purified protein or to install PTM by chemical modification after a protein is synthesized, which make it difficult to study the function of PTM in vitro. Native chemical ligation allows complete modification of a given site in a protein, but is often restricted by the length of the protein, and cannot be used in in vivo study. Incorporating unnatural amino acids to recapitulate or mimic such PTM could generate proteins with fully modified residues at a particular site, allowing both in vivo and in vitro study.30-31 In addition to being an important epigenetics feature, PTMs also appear in many enzymes, tuning the structure and activity. A prominent PTM is the His-Tyr cross-link in hemecopper oxidases (HCOs), which are terminal oxidases in the respiration chain. They are responsible for 90% of oxygen consumption by biological reactions on earth.32 Since HCOs can catalyze oxygen reduction with high efficiency and speed,33-34 it is interesting to design an artificial enzyme with such features, especially for application in an enzymatic fuel cell. In HCOs, there is a pair of covalently attached His and Tyr residues in the active site – the binuclear center. Such a linkage is formed during the first catalytic turnovers. The suggested function is to tune the pKa of the tyrosine or correctly position the motif. After studying the HisTyr cross-link motif and mimicking it in a small molecule model complex,35-36 installing the feature into a model protein is crucial for further studies mimicking and investigating the oxidase activity. Liu et al. used 2-amino-3-(4-hydroxy-3-(1H-imidazol-1-yl)phenyl)propanoic acid (imiTyr 1) to mimic the native Tyr-His cross-link.37 imiTyr was incorporated into myoglobin through genetic code expansion. Together with other mutations, the myoglobin-based model (imiTyrCuBMb) recapitulated the structural features of the HCOs. As a result, imiTyrCuBMb


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exhibited an oxygen consumption rate of 2.2 min-1, with less than 6% converted to reactive oxygen species (ROS). Another example of active site mimicking is a Tyr-Cys cross-link. This cross-link is between the C3 ring carbon of Tyr and Sγ of Cys and appears in natural enzymes, including galactose oxidase, glyoxal oxidase, cysteine dioxygenase, sulfite reductase and cytochrome c nitrite reductase. The model compound studied indicated that the cross-link lowers the pKa and reduction potential of Tyr, facilitating enzymatic reactions.38 To probe such PTMs in proteins, Wang and coworkers synthesized 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (3-Methylthiotyrosine or MtTyr 2). 39 They introduced MtTyr to the 33rd position of myoglobin through genetic code expansion to mimic the active site of cytochrome c nitrite reductase, including a heme and Tyr-Cys cross-link. The designed protein reduced hydroxylamine, an intermediate in nitrite reduction, at a rate of 800 min-1. The increased hydroxylamine reductase activity of the Mb mutant with MtTyr suggested that MtTyr can mimic the Tyr-Cys cross-link in a model protein. The two cases showed that unnatural amino acids can mimic PTMs in the active sites of native enzymes. 2.3 Unnatural amino acid for activity tuning In the active site of enzymes, there are key residues involved in the reaction. Unnatural amino acids analogous to the natural ones can be used to keep the structure of the active site unperturbed, while altering certain properties crucial for the catalytic reaction. Tyr is a redox-active amino acid present in many enzymes, where it is either directly involved in catalysis or relays electrons and/or protons to the active site. Two properties, the acid dissociation constant (pKa) and the redox potential of the phenol group, are directly related to the


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function of Tyr in catalysis. Historically, the TyrRS/tRNACUA pair from the archaebacteria Methanocaldococcus jannaschii was the first orthogonal aaRS/tRNA pair used to introduce an unnatural amino acid into a protein.28 Many Tyr analogs with different ring substitutions have been incorporated into proteins. Notably, o-substituted tyrosines (o-Cl, Br, I, NH2, OH, NO2) and fluorotyrosines (2,3,5-, 2,4,5-, etc.) are structurally similar to the native Tyr, but the pKa values and redox potentials span a wide range (Table 1). The substitution of Tyr with FnY (3,5difluorotyrosine, 2,3- difluorotyrosine, 2,3,5- trifluorotyrosine, and 2,3,6- trifluorotyrosine) demonstrated minimal structural perturbation to ribonucleotide reductase.40 These unnatural amino acids are useful tools to tune the activity of native enzymes, including ribonucleotide reductase41 and ketosteroid isomerase.42 Table 1. pKa values and reduction potentials of Tyr analogs Amino acid

pKa

Reduction potential Ep (Y·/Y-, mV vs NHE) b in α3Y (pH 5.5) a

Tyr

9.8 43

1065

642 44

3-fluorotyrosine 3

8.4 44

-

705 44

3,5-difluorotyrosine 4

7.2 44

1040

755 44

2,3-difluorotyrosine 5

7.7 44

1136

810 44

2,3,5-trifluorotyrosine 6

6.4 44

1104

853 44

2,3,6-trifluorotyrosine 7

6.9 44

1200

911 44

3-chlorotyrosine 8

8.1 43

-

734 43

3,5-dichlorotyrosine 9

6.3 43

-

808 43

3-methoxytyrosine 10

9.9 45

-

480 45

a

formal reduction potential measured by square wave voltammetry.

b

peak potential measured by deep potential voltammetry or cyclic voltammetry


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In addition to the MjTyrRS/tRNA, E. coli TyrRS/tRNA,46 LeuRS/tRNA, 47 TrpRS/tRNA,48 Methanosarcina barkeri and Methanosarcina mazei PyrrolysylRS RS/tRNA pairs49 are also used in genetic code expansion, allowing incorporation of unnatural amino acids analogous to Lys, His, Ser and Cys. These unnatural amino acids can be used to replace corresponding native amino acids in the active site, tuning enzymatic activities. For example, the proximal histidine of ascorbate peroxidase was replaced by Nδ-methyl histidine (11) to break a conserved Asp-His hydrogen bond.50 The modified enzyme showed a 4-fold increase of total turnover number and similar catalytic efficiency compared to the WT. The method can also be applied in artificial metalloenzymes. The active site Tyr is proposed to provide an electron and a proton during the oxygen reduction reaction in hemecopper oxidases.32 To probe the exact role of the Tyr, unnatural amino acids analogous to Tyr are introduced into the aforementioned Mb-based oxidase model (Figure 3). The oxidase activity of the enzyme is correlated with the pKa value and redox potential of the Tyr or Tyr analog at the active site, suggesting an active role of Tyr in the oxidase reaction.43,45

Figure 3. Unnatural amino acids used to probe the role of Tyr in an artificial oxidase.


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2.4 Site-specific enzyme immobilization by unnatural amino acid incorporation Enzyme immobilization is the attachment of enzyme to a material (matrix/support) that is different from that for substrates and products51, which can remarkably improve the longevity and reproducibility of an enzyme during bio-catalytic applications. The development of this technique continuously impacts the field of bio-catalysis-based processes and bioelectronics research. Site-specific unnatural amino acid incorporation, in this scenario, offers a great opportunity to enable rationally engineered proteins to be directly and site-specifically immobilized onto surfaces. Smith et al. site-specifically incorporated a uniquely reactive unnatural amino acid p-propargyloxyphenylalanine (12) at a specific location in green fluorescent protein (GFP) by cell-free protein synthesis52. The modified GFP was then directly and covalently attached to super-paramagnetic beads by the unnatural amino acid in a single click reaction. The immobilized GFP retained activity and stability under harsh conditions, including freeze-thaw cycling and incubation in urea at elevated temperatures. The enhanced stability of the immobilized protein compared to unattached protein suggested that this is a promising strategy for enzyme immobilization. In addition, Seo et al. employed the genetic code expansion and click chemistry methods for controlled and oriented immobilization of the pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 in Homo sapiens.53 Kinetic analysis of ligand binding using surface plasmon resonance (SPR) revealed that oriented immobilization of site-specifically biotinylated DrrA (DrrA-AzF-Bio, 13) resulted in an approximately 10-fold higher binding affinity compared to the immobilization of randomly biotinylated DrrA (DrrAran-Bio). Undoubtedly, the rational incorporation of unnatural amino acid offers a powerful way of site-specific coupling of a protein to material. Especially for


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nanoscale materials, it could be very useful in establishing a bio-hybrid nano device. For example, Yoshimura et al. utilized a modified Staudinger–Bertozzi ligation to couple a carbon nanotube end with an azide group which was site-specifically incorporated into a Ca2+ sensor protein, calmodulin, without affecting calcium-dependent substrate binding.54 In a bio-electrochemical system, electron transfer between the catalytic center of the enzyme and the electrode occurs through either direct electron transfer (DET) or mediated electron transfer (MET). Thus, enzyme immobilization on the electrode can occur by wiring enzymes to the electrode. In this area, a great deal of effort has been devoted to engineering both enzymes and electrodes with a common goal: improving the electron transfer efficiency between enzymes and electrodes while maintaining enzymatic activity and stability. This goal seeks to generate biosensors with better sensitivity/selectivity and biofuel cells with higher power outputs and/or open circuit potentials. Site-specific unnatural amino acid incorporation has emerged as a robust approach for wiring enzymes to electrodes in a delicately controlled manner. The optimization of a specific site on enzyme as an anchor point to the electrode can minimize the loss of activity/stability during immobilization and also provide precise control of enzyme orientation on electrode. Moreover, the variety of unnatural amino acids incorporated enables a wide range of bio-conjugated approaches that can be employed for wiring the enzyme either directly to various electrode materials or with the help of rationally designed surface modification linker molecules. For example, Amir et al. demonstrated the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF, 13) in alcohol dehydrogenase and displayed it on living bacteria.55 To facilitate electron transfer between the enzyme and a gold electrode, a linker containing an alkyne and thiol moiety on opposite ends was synthesized and site-specifically


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attached to the dehydrogenase through a copper(I)-catalyzed azide−alkyne cycloaddition reaction.55 Similarly, an unnatural amino acid, AzF (13), was incorporated into specific sites of a small laccase from Streptomyces coelicolor (SLAC). A heterobifunctional crosslinker, cyclooctynyloxyethyl 1-pyrenebutyrate (PBCO), was synthesized and used to functionalize MWCNTs on buckypaper via π−π stacking. Directional immobilization of SLAC on the buckypaper was achieved by a copper-free click reaction between AzF on the enzyme and cyclooctyne on the MWCNT.56 An improved DET process was observed after optimization of the tethering site. 3 Non-native cofactors for artificial metalloenzyme design Cofactors or prosthetic groups, many of which are metal ions or contain metals, are the basis for catalysis in metalloenzymes. Through intricate biosynthesis, nature can assemble these metal cofactors, some with complex chemical structures, and transfer them into the host protein for function. Compared to the already rich native cofactor repertoire, chemists can then synthesize a large variety of metal complexes with different types of metal, difficult-tosynthesize ligands, and novel chemical structure. The incorporation of non-native cofactors greatly expands the reactions catalyzed by metalloenzymes. Compared to homogeneous catalysis by metal complexes, embedding metal complexes in a host protein provides better control of the coordination environment, especially in the second coordination sphere,57 making it possible to achieve better regio-, stereo-selective catalysis. There are many excellent reviews in this field8,14,16,58-79, including a recent review by Lewis and Ward,80 which summarized different chemistry performed by artificial metalloenzymes. In the following section, we will discuss


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methods to incorporate non-native cofactors into proteins, with an emphasis on the genetic code expansion strategy. 3.1 Incorporation of non-native cofactors through covalent interactions Non-native cofactors can be inserted or assembled in situ in the host protein in a nonspecific manner.81-82 Having a defined site for the non-native cofactors ensures full occupancy during catalysis due to high affinity and also makes it possible to further manipulate the protein for improved catalytic performance. Native cofactors, such as different types of hemes, can be either covalently or non-covalently attached to the protein. Non-native cofactors can be readily attached to a protein with different bioconjugation methods. Nitrobindin (Nb) is a β barrel protein with a large cavity for heme binding. After introducing a Cys in the cavity of Nb, various non-native cofactors with the maleimide moiety are covalently attached to the apo-protein (Figure 4). Hayashi and coworkers demonstrated that Nb with different cofactors can perform reactions such as hydrogenation,83 ring-closing metathesis,84 phenylacetylene polymerization,85 benzylic oxidation86 and Diels–Alder reaction.87


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Figure 4. Crystal structure of mutant nitrobindin M75L/H76L/Q96C/M148L/H158L covalently linked with [Rh(Cp-Mal)(COD)] (NB4-Rh). Mutated residues are in red, and non-native cofactor is in cyan. PDB ID:3WJC. Metal-salen and salophen cofactors have similar structures to heme. They can readily be incorporated into certain heme proteins. Lu and coworkers later developed a dual anchoring strategy to attach a Mn-salen cofactor through two Cys on myoglobin.88 Myoglobin with dualanchored Mn-salen demonstrated 51.3% ee for sulfoxidation of thioanisole, a significant increase compared to non-covalent or single-anchored metalloenzyme. They further optimized the system by mutating the host protein by altering the sites of cofactor anchoring.89-90 3.2 Incorporation of non-native cofactors with non-covalent interactions


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Non-native cofactor incorporation with non-covalent interactions is more challenging, since more protein-cofactor interactions are needed to achieve high affinity. In the simplest scenario, the native metal ion in a metalloenzyme is replaced by a non-native one. Heme proteins, especially cyt P450s, are known for oxene transfer during catalysis. Arnold and coworkers generated a series of mutations of P450BM3. By tuning heme ligands and other residues, the P450BM3 mutant can catalyze cyclopropanation of styrenes via putative carbene transfer.91 Arnold, Fasan and coworkers engineered different heme proteins, keeping the heme cofactor, to perform carbene Si–H and C–H insertions, Doyle–Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion.92 Complementary to the protein mutations, replacing heme with non-native cofactors expands the activity of heme enzymes. Cofactor substitution in heme proteins can be achieved through different strategies. The methods for non-native heme incorporation can also be references for cofactor substitution in other types of proteins. Traditionally, apo protein is directly purified or prepared by denaturing the holo heme protein through acid treatment and extracting heme with organic solvents. The protein is then reconstituted in presence of non-native cofactors. Watanabe and coworkers developed a method to reconstitute heme protein in cell lysate, avoiding irreversible denaturation of heme proteins in heme extraction.93 To enable in vivo non-native cofactor incorporation, Jasanoff and coworkers overexpressed a native heme-transport channel, ChuA, in E. coli.94 This strain allows import of metal-swapped protoporphyrin IX derivatives, and insertion of these non-native cofactors into heme protein. The efficiency of the process can be further improved by using iron-deficient


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minimal media to minimize heme biosynthesis. A manganese porphyrin substituted P450BM3 was prepared in this strain and was used as a MRI contrast agent. Marletta and coworkers constructed an E. coli strain, RP523, with an essential gene for heme biosynthesis knocked out and an unknown mutation to render the cell heme-permeable.95 In this strain, a ruthenium porphyrin substituted H-NOX was prepared as an O2 sensor. In vivo incorporation of non-native cofactors faces competition by the native heme. For example, ~2-5% of protein from ChuA-overexpression strain contains heme rather than the nonnative heme.94 To overcome this problem, Brustad and coworkers engineered P450BM3 by making mutations T269V/L272W/L322I/ A406S (WVIS) to increase its selectivity to irondeuteroporphyrin IX (Fe-DPIX). The P450BM3 mutant and Fe-DPIX pair are selective to each other, but not to other native protein or heme, thus forming an orthogonal protein/cofactor pair. 96 These strategies have been used in myoglobin and different cyt P450s to construct artificial metalloenzymes for better or new activities. Hartwig and coworkers replaced Fe in FePPIX (Fe-protoporphyrin IX or b-type heme) in myoglobin with eight different transition metals. The affinity of Mb to the non-native cofactors was comparable to the native state, since only the transition metal was changed. Mb with Ir(Me)-PPIX were active toward inactivated olefins and diazoesters, with enantio- and diastereoselectivity, which was not observed in engineered heme proteins.97 They applied the same Ir(Me)-PPIX to a thermostable cyt P450, CYP119, in combination with mutations of the protein, and the metalloenzyme exhibited 98% ee, 35,000 TON, and 2550 hours−1 TOF in the carbene transfer reaction (Figure 5).98 The performance of the metalloenzyme in a novel reaction was comparable to that of the native enzyme in the corresponding native reaction. The same enzyme, Ir(Me)-PPIX CYP119, was also shown to catalyze nitrene transfer in C–H-bond amination reaction with high selectivity.99 Fasan and


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coworkers prepared myoglobin with non-native hemes from a ChuA overexpression E. coli strain.100 They systematically studied carbene transfer reactions (cyclopropanation, N-H insertion, and S-H insertion) by myoglobin with Rh, Ru, Ir, Co and Mn porphyrins. Catalytic activity of the articial metalloenzyme is affected by the nature of the metal center, its oxidation state and its first-sphere coordination environment. Myoglobin with Co- or Mn-porphyrins are able to catalyze carbene C–H insertion reaction involving phthalan and ethyl a-diazoacetate, a reaction inaccessible by the native myoglobin. Brustad and coworkers prepared P450BM3 with Ir(Me)-deuteroporphyrin IX using a previously developed orthogonal protein/heme pair.101 The artificial metalloenzyme showed enhanced activity for aliphatic and electron-deficient olefins in olefin cyclopropanation reaction, compared to the native P450BM3.

Figure 5. Crystal structure of wild type CYP119 (PDB ID: 1IO7) and carbene transfer reactions catalyzed by Ir(Me)-PPIX substituted CYP119 mutants. In the crystal structure, Fe-PPIX is in cyan, and the residues screened in directed evolution are in red. In addition to protoporphyrin IX with different metals, other metallocycles with similar structure to heme can also be incorporated into heme proteins. Watanabe and coworkers


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incorporated a Cr(III)(salophen) moiety into apo myoglobin.102 The artificial enzyme catalyzes an enantioselective sulfoxidation reactiofn with up to 13% ee and up to 83×10-3 min-1 turnover. The same group incorporated Rhodium 2,6-bis(2-oxazolinyl)phenyl into apo myoglobin.103 The metal complex is positioned in the cavity of myoglobin with an almost perpendicular arrangement to that of the heme. Fasan and coworkers replaced the native heme in myoglobin with an iron-chlorin e6 complex.104 The catalyst was able to catalyze the cyclopropanation of vinylarenes with a turnover number of 6970, a turnover rate of 2000/min, and high stereoselectivity (up to 99% de and ee). Hayashi and coworkers incorporated iron-porphycene into myoglobin. Due to the strong ligand field of porphycene and fewer intersystem crossing steps, formation of carbene intermediate is accelerated in the artificial metalloenzyme, resulted in a 26-fold increase of kcat/Km compared to the native myoglobin.105 Non-native cofactors can be rationally designed in small proteins or peptides. Pioneer studies in this area were initiated with de novo design of heme binding peptides. Suslick and coworkers synthesized a series of cyclic peptides to bind Fe(III)/Co(III) porphyrins.106 They discovered neutral and positively charged histidine-containing peptides had high affinity to metalloporphyrin. In addition, CD experiments confirmed that the metalloporphyrin bound to peptides with high affinity and isodichroic behavior. In a subsequent study, a peptide-ruthenium porphyrin complex was designed and synthesized.107 NMR experiments suggested that the peptide formed a helix structure when bound to ruthenium porphyrin. Alternatively, Degrado and coworkers designed and synthesized a small protein with a four-helix bundle that self-assembled with an unnatural diphenylporphyrin108. In another study, they reported a membrane protein with two porphyrins coordinated that was designed de novo.109 Such attempts would benefit the de novo design of novel heme protein scaffolds in future studies.


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In addition to heme proteins, other cofactor binding proteins can be rationally designed. Degrado and coworkers used a minimal scaffold with four α helices as the starting point. By retro-analysis and computer algorithms, they designed a di-iron center in the protein.110 The due ferri protein designed de novo was further engineered for oxidation reactions.111-114 High affinity non-covalent interaction between a protein and a non-native cofactor can be obtained by screening or computational design. Antibodies can recognize small molecule haptens with high affinity, and antibody selection and affinity improving methods have been well established.115 Schultz and coworkers first reported a monoclonal antibody for N-methylmesoporphyrin IX.116 The antibody with Fe(III)-mesoporphyrin, generally called hemoabzymes, exhibited peroxidase activity. Many groups, including Kawamura-Konishi, Imanaka, and Mahy further selected antibodies with peroxidase activity.75 In addition to screening, computational design can also improve protein-cofactor affinity. Baker, Ward and coworkers used Rosetta design to identify mutations that increase the affinity between human carbonic anhydrase II (hCAII) and (η5-Cp*)Ir(pico)Cl up to 64 times (Figure 6).117 The activity and enantioselectivity of the artificial transfer hydrogenase for the production of (S)-salsolidine were also increased.


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Figure 6. Computational design to increase cofactor affinity. Reprinted from Figure 1 of Ref 117 with permission from the American Chemical Society. 3.3 Incorporation of non-native cofactors through supramolecular anchoring It is challenging to design or screen for a host protein with high affinity for a non-native cofactor. Even if such a protein exists, it is laborious, if not impossible, to design or screen host proteins for each new non-native cofactor. In other cases, a “Trojan horse” strategy,118 or supramolecular incorporation, is used to ensure tight binding between the host protein and the non-native cofactor. The metal complex responsible for catalysis is covalently linked to another molecule, which can bind to the host protein with high affinity. Mahy and coworkers designed a hemoabzyme with peroxidase activity.119-120 Instead of selecting the antibody for heme, they used an existing antibody for estradiol, and the Feporphyrin was covalently linked to estradiol. This strategy has also been used in other proteinsmall molecule systems, such as HSA-ibuprofen121 and antibody-testosterone.122


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Biotin-streptavidin (Sav) is one of the strongest non-covalent interactions in nature, with a Kd of ≈10−14 mol/L.123 Wilson and Whitesides pioneered in using Biotin-Sav for asymmetric hydrogenation reactions.124 Ward and coworkers successfully used a biotin moiety to anchor a range of metal complexes into Sav, creating artificial metalloenzymes for imine reduction,125 ketone reduction, 126 metathesis, 127-128 C–H activation,129 sulfoxidation, and so on (Figure 7 and Figure 8). The success of biotin-streptavidin technology points out some general rules for metalloenzyme design through supramolecular incorporation. First, the host protein should be stable enough for future mutations and reactions in water-organic solvent mixtures. Second, the host protein and corresponding native cofactor should have high affinity. Finally, the host protein should have a suitable cavity for different non-native cofactors.

Figure 7. Artificial metalloenzymes based on the biotin-(strep)avidin technology. Reprinted from Figure 2 of ref130 with permission of the American Chemical Society.


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Figure 8. Reactions catalyzed by artificial metalloenzymes based on the biotin-(strep)avidin supramolecular incorporation. Adopted from ref74 with permission of the American Chemical Society. 4 Unnatural amino acid for anchoring cofactors Unnatural amino acids can bind metal ions in a bidentate fashion with high affinity, or they can anchor cofactors through bio-orthogonal reactions, making it plausible to design a new cofactor binding site. Unnatural amino acids with bipyridine, hydroxyquinoline or pyrazolylphenol groups as side chains (14, 15, 16, 17) have been incorporated into proteins. The metal affinity of these unnatural amino acids is orders of magnitude higher than natural amino acids. In the case of 2-amino-3-(8-hydroxyquinolin-5-yl)propanoic acid (HqAla, 17), the unnatural amino acid has a KD of 0.1 fM for Cu(II), which is tighter than many native copper sites. The high metal affinity of these unnatural amino acids removes the need to design other ligands to further increase the affinity of the site, dramatically decreasing the workload in designing a metal site. Bipyridylalanine (BpyAla, 14) was the first bidentate amino acid ligand introduced into protein.131 E. coli catabolite activator proteins (CAP) are known bind double-stranded DNA in a sequence-specific manor. Schultz and coworkers introduced BpyAla into a CAP protein. After unnatural amino acid insertion, the protein could still bind DNA, and in the presence of Cu(II) and reductant, the protein cleaved the bound dsDNA.132 Roelfes and coworkers introduced BpyAla at the dimer interface of Lactoccocal multidrug resistance regulator (LmrR, Figure 9).133 BpyAla binds Cu(II) inside the protein. Upon addition of Cu(II) ions, the protein catalyzes Friedel–Crafts alkylation reaction with up to 83% ee and 94% conversion of 1 mM substrate


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over 3 days. A combination of cluster model calculations (QM), protein–ligand docking and molecular dynamics simulations were used to design a metallo-hydratase in silico with LmrR as the scaffold and BpyAla as the Cu(II) anchor.134 The artificial metalloenzyme was able to catalyze the hydration of α,β-unsaturated 2-acyl pyridine with up to 64% ee, in agreement with computational predictions. Unnatural amino acids can selectively anchor metal ions and also anchor non-native cofactors, as we will discuss in the following sections. Such selective anchoring is important for designing a functional catalytic site.

Figure 9. Design of Cu(II) binding site with BpyAla in LmrR. Protein catalyzes the Friedel– Crafts reaction. Reprinted from ref133 with permission from the Royal Society of Chemistry. Larger non-native cofactors can be introduced into the protein using unnatural amino acids with bio-orthogonal reaction groups. Lewis and coworkers introduced 4-azidophenylalanine (AzF, 13) into model proteins tHisF and phytase, and they demonstrated the incorporation of the cofactors (Rh2-tetraacetate and Mn- and Cu-terpyridine complexes) with


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strain-promoted azide-alkyne cycloaddition (SPAAC, Figure 10).135 They used the same strategy to introduce AzF into the cavity of a thermostable prolyl oligopeptidase and to anchor a dirhodium tetracarboxylate complex, followed by mutations to expand cavity size.136 The engineered protein is able to catalyze enantioselective cyclopropanation of a broad range of olefins. Installing a metal complex in a host protein not only reduces by-product formation but also facilitates the reaction in aqueous solution.

Figure 10. Cofactor anchoring by bio-orthogonal reactions. Reprinted from Figure 1 of ref135 with permission from Wiley-VCH.

5 Methods for metalloenzyme design with unnatural amino acids and non-native cofactors Compared to mutagenesis of natural amino acids, experimental incorporation of unnatural amino acids requires the addition of unnatural amino acid into the growth medium, increasing the cost of experiment. In most cases, only one kind of unnatural amino acid can be incorporated into a protein at a single site. In computational studies, force field parameters for unnatural amino acids and non-native cofactors are lacking. These differences hinder the


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application of protein engineering with unnatural amino acids and non-native cofactors, and most protein design and engineering are based on semi-rational design. There are several examples of research applying existing methods for protein engineering with unnatural amino acids and non-native cofactors. Phage display is a common method where the target protein is displayed on the surface of bacteria phage and then subjected to screening and manipulation. Unlike with natural amino acids, screening an unnatural amino acid library requires mutating the gene with TAG and supplementing medium with the corresponding unnatural amino acid. Schultz and coworkers screened for an Fe-binding peptide with BpyAla by phage display.137 Hartwig and coworkers applied a directed evolution strategy on Ir(Me)-PPIX Mb and successfully increased the selectivity of the artificial metalloenzyme for either enantiomers (Figure 11).97


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Figure 11. Directed evolution strategy for increasing the enantioselectivity of a metalloenzyme with a non-native cofactor. Reprinted from Figure 4 of ref97 with permission from Springer Nature. Rosetta is a computational software suite used for de novo design of many enzymes. Unfortunately, parameterization of unnatural amino acids and non-native cofactors is lacking in Rosetta. Baker and coworkers parameterized BpyAla, a metal chelating unnatural amino acid with quantum chemistry calculations. A metal coordinating site with BpyAla was constructed and installed in a host protein using Rosetta, and similar to proteins with natural amino acids, the


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experimentally determined structures matched the designed ones (Figure 12).138 Their work demonstrates the ability of Rosetta to design protein with unnatural amino acids.

Figure 12. Metalloprotein designed by Rosetta. Reprinted from Figures 2 and 4 of ref138 with permission from the American Chemical Society. Liu and coworkers realized the incorporation of two types of unnatural amino acids into a single protein by suppressing the amber (UAG) and ochre (UAA) stop codons at the same time.139 Wang and coworkers introduced a release factor 2 with increased efficiency toward UAG codon and evolved E. coli that survives without release factor 1 (RF1).140 With RF1 knockout, the efficiency of unnatural amino acid incorporation increased significantly, so that the same unnatural amino acid could be incorporated at multiple sites within a protein. With the


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increasing power of synthetic biology, artificial genomes or deeply engineered genomes are possible. Isaacs et al. developed a method for genome-wide codon replacement.141 Church, Isaacs and coworkers used this tool to first replace all UAG stop codons in the E. coli genome with unnatural amino acid, and then they further modified the genome to eliminate another 12 codons.142-143 The E. coli mutants with genome-wide UAG codon removal demonstrated a higher efficiency for unnatural amino acid incorporation and resistance to T7 phage. Romesberg also introduced an unnatural base pair into the cell, expanding the genetic alphabet.144 The expanded genetic alphabet provides more than a dozen empty codons.145 In the near future, it is likely that there will be an engineered organism that can integrate many different unnatural amino acids into multiple sites in a protein, enabling enzyme engineering with genetically encoded unnatural amino acids to be a more common practice. In addition to the restrictions posed by the methodology, unnatural amino acids must be chemically synthesized and added to the growth medium, which is laborious and expensive. Phillips and coworkers developed a method to use an enzyme, tyrosine phenol lyase (TPL), to convert substituted phenols into L-amino acids.146 Stubbe and coworkers then applied the method to synthesize fluorinated Tyr and incorporated them into proteins.44 To further expand the substrate scope of TPL, Wang and coworkers mutated TPL to catalyze the conversion of more complex unnatural amino acids.147 Enzymatic conversion of unnatural amino acids makes it possible for a biochemistry lab without strong synthetic chemistry expertise to carry out unnatural amino acid incorporation. The next step for unnatural amino acid synthesis is to integrate the entire biosynthesis pathway in the cell. Schultz and coworkers constructed an artificial biosynthesis pathway for paminophenylalanine (pAF, 18) in E. coli by introducing three genes from Streptomyces


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venezuelae (Figure 13).148 The engineered bacteria could synthesize pAF from basic carbon sources and incorporate the unnatural amino acid into protein in presence of the corresponding aaRS and tRNA. Similarly, Pyrrolysine and derivatives, and S-allylcysteine (Sac) can be biosynthesized from simple precursors, such as D-ornithine and derivatives, and ally mercaptan and o-acetylserine.149-151 This strategy will significantly reduce the cost for unnatural amino acid incorporation into proteins.

Figure 13. Biosynthesis of p-aminophenylalanine in E. coli by heterologous gene expression. Reprinted from ref 148 with permission from the American Chemical Society. 6 Conclusion and perspectives Unnatural amino acids have been used in enzyme design by mimicking the active site, especially the PTM; tuning the activity of the enzymes; as an anchor to harbor metal ions for catalysis; or in bio-orthogonal chemistry to immobilize an enzyme on an electrode. The incorporation of non-native cofactors can be achieved with non-covalent interactions or covalent attachment to expand the scope of existing metalloenzymes. Compared to natural amino acids and cofactors, unnatural amino acids and non-native cofactors provide unique advantages in enzyme design. They enable the integration of unprecedented structures into proteins for novel


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reactions or substrates, and they provide tools for activity tuning, for example, with a series of unnatural amino acids with similar structures but different pKa values or reduction potentials. Compared to large number of the existing genetically encodable unnatural amino acids, only a fraction have been used in enzyme design. The current unnatural amino acid pool provides us with rich resources to exploit enzyme design. The authors consider the following directions promising in generating the next breakthrough in this field. Chemists have developed many potent small molecule catalysts. Comparing to small molecule catalyst, enzymes are better in stero- and regio- specific reactions. Many groups have endeavored in combining small molecule catalyst with protein scaffold and the history has been reviewed by Ward and Lewis recently.80 Previous efforts to attach a small molecule to the protein have often been restricted by the availability of functional groups and specificity. As demonstrated by Lewis and others,135 combining unnatural amino acids with bio-orthogonal reactivity and non-native cofactors could provide more tools for enzyme design. Thanks to the development in photovoltaics, electricity is becoming a promising source of clean and sustainable energy. Electrocatalysis has gained much attention recently. For enzyme catalysis, unnatural amino acids could be used in site-specific immobilization for improving electron transfer efficiency and enzyme performance.55 Meanwhile, non-native cofactors, such as a modified hemin cofactor bearing an alkyne group can be used to anchor protein to the electrode for electrocatalysis.152


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Overall, unnatural amino acids and non-native cofactors are promising tools for enzyme design. With the accumulation of genetically encodable unnatural amino acids and non-native cofactors, our ability to rationally design protein catalyst will be significantly improved.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We are grateful for the financial support from the National Key Research and Development Program of China under Award 2016YFA0501502, the National Science Foundation of China under award 91313301, 21325211, 31500641. ABBREVIATIONS PTM, post-translational modifications; KSI, ketosteroid isomerase; DET direct electron transfer; CNT, carbon nanotube; GFP, green fluorescence protein; SPR, surface plasmon resonance. REFERENCES (1) (2)

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Artificial Metalloenzyme Design with Unnatural Amino Acids and Non

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