Bioconjugation and Active Site Design of Enzymes Using Non-natural

Review pubs.acs.org/IECR

Bioconjugation and Active Site Design of Enzymes Using Non-natural Amino Acids Inchan Kwon*,†,‡ and Byungseop Yang† †

School of Materials Science and Engineering (SMSE) and ‡Department of Biomedical Science and Engineering (BMSE), Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea ABSTRACT: Enzymes are biocatalysts that play key roles in diverse chemical reactions in living organisms and industrial conversion processes generating value-added products. Since wild-type enzymes obtained from nature are normally not optimal for various applications, enzyme engineering is usually required for enhanced or new properties. Site-specific incorporation of a non-natural amino acid became a powerful protein-engineering tool. In this short review, we briefly summarize our contribution to enzyme complex formation and active site design of enzymes using the technique of site-specific incorporation of a nonnatural amino acid. First, site-specific incorporation of a non-natural amino acid at a permissive site of an enzyme led to bioconjugation to other molecules without compromising critical properties. Murine dihydrofolate reductase (mDHFR) was site-specifically conjugated to a biotin via click chemistry to achieve site-specific immobilization. Similarly, formate dehydrogenase (FDH) was site-specifically conjugated to an organometallic catalyst for cofactor regeneration. Furthermore, successive applications of two different click chemistries allowed site-specific coupling of FDH and mannitol dehydrogenase for the enhanced overall reaction efficiencies via substrate channeling effects and active site-orientation control. Besides enzyme bioconjugation, precise control of non-natural amino acid incorporation also allows for active site modification with a non-natural amino acid. Introduction of a bulky nonnatural amino acid into the mDHFR active site lowered binding affinity to its inhibitor methotrexate without compromising binding affinity to its substrate dihydrofolate. Similarly, introduction of a bulky non-natural amino acid into the mDHFR active site led to the alteration of substrate specificity toward a poor substrate folate over a good substrate dihydrofolate.

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experimentally test with restricted time and labor.8−10 These days, researchers are seeking a new strategy to further expand the amino acid sequence space. Along with several other groups, we have been investigating expansion of the amino acid sequence space for enzyme engineering using non-natural amino acids.11−15 In nature, almost all proteins, including enzymes, consist of only 20 amino acids. We hypothesized that expansion of the number of amino acids available for enzyme synthesis would allow design/screening of novel enzymes beyond the limits set by nature. Since general strategies of nonnatural amino acid incorporation into enzymes were discussed in detail in other reviews,16−19 herein we briefly explained the site-specific incorporation technique of non-natural amino acids into enzymes. Then, we focused on summarizing our contribution to two topics, forming enzyme complexes via bioconjugation and engineering of the enzyme active site using non-natural amino acids, which was not extensively handled in other reviews. For enzyme biconjugation, the reactivity of a non-natural amino acid side chain played an important role, whereas other properties such as shape, size, and polarity of the

INTRODUCTION Enzymes play key roles in maintaining life in living organisms. Enzymes catalyze diverse biochemical conversion processes under mild conditions. Because of their catalytic properties, enzymes are considered biocatalysts. Biocatalysts have been used to produce pharmaceutical intermediates and food ingredients. Recently, enzymes have been intensively investigated for biofuel synthesis. However, industrial uses of enzymes are quite restricted due to several issues. Besides a moderate stability issue, wild-type enzymes usually do not have desirable catalytic properties for specific applications, and so require enzyme engineering. Enzyme-engineering strategies were evolved to expand amino acid sequence space.1 Initially, site-directed mutagenesis was developed to mutate a single site at once. The success of site-directed mutagenesis greatly depends on the structural information and/or biochemical properties of a target enzyme, which are not always available.2−4 To overcome this limitation, directed evolution was developed.5−7 Multiple mutations were introduced into random sites of a target enzyme, resulting in a large number of mutant libraries, which are subjected to high-throughput screening. Multiple rounds of library construction and screening led to enzymes with enhanced properties. Recently, computational design and in silico screening were developed to expand the amino acid sequence space beyond the limit of which we can © 2017 American Chemical Society

Received: Revised: Accepted: Published: 6535

February 13, 2017 April 3, 2017 May 18, 2017 May 18, 2017 DOI: 10.1021/acs.iecr.7b00612 Ind. Eng. Chem. Res. 2017, 56, 6535−6547

Review

Industrial & Engineering Chemistry Research

Figure 2. Enzymatic activity of the mDHFR after site-specific biotinylation and immobilization. (a) Activity of the mDHFR-43biotin versus the mDHFR-43pEthF. Error bars represent standard errors (n = 3). (b) Activity of the immobilized mDHFR-43biotin. Streptavidincoated wells were incubated with 50 μL of the mDHFR-43biotin and the mDHFR-43pEthF at 1 mg/mL, separately, for 30 min at RT. After washing, the enzymatic reaction was initiated at 25 °C by adding 200 μL of assay buffer, and monitored by spectrometry. Error bars represent standard errors (n = 3). Reproduced with permission from ref 47. Copyright 2014 Public Library of Science.

Figure 1. Effect of various reductants on CuAAC reaction rates and activities of the mDHFR-43pEthF and the mDHFR-179pEthF. (a) Time course of CuAAC reactions initiated by ascorbate, TCEP, and DTT. Reactions were performed at 25 °C by adding 2 mM reductant to a phosphate-buffered (pH 8.0) mixture containing 30 μM of the mDHFR-43pEthF (black) or the mDHFR-179pEthF (gray), 60 μM of azidocoumarin, 1 mM of CuSO4, 1 mM THPTA. Fluorescence evolution was recorded at λex = 400 nm and λem = 470 nm. (b) Relative loss of enzymatic activity after incubation with various CuAAC systems in the absence of azidocoumarin (1−4). Incubation times were 15 min for system 1 and 4, 12 h for system 2, and 2 h for system 3. Activity losses were normalized to that in system 1. Error bars represent standard errors (n = 3). Two-sided Student’s t tests were applied to the data ((∗) P < 0.05). (c) Effect of ascorbate-driven dye labeling of the mDHFR-43pEthF on the enzymatic activity. The labeling was performed at 25 °C for 15 min in the presence of 30 μM of the mDHFR-43pEthF, 60 μM of azidocoumarin, 1 mM of CuSO4, 1 mM THPTA, and 2 mM ascorbate. Error bars represent standard errors (n = 3). Reproduced with permission from ref 47. Copyright 2014 Public Library of Science.

multiple-site-specific incorporation. First, researchers have to choose a non-natural amino acid suitable for our purpose. So far, more than 150 non-natural amino acids have been shown to be able to be introduced into a protein in vivo. Considering the size, polarity, and reactive functional group of a side chain, researchers need to choose a desirable nonnatural amino acid. Second, researchers have to express a heterologous orthogonal pair of tRNA/synthetase specific for a non-natural amino acid in expression hosts. Several orthogonal pairs derived from Methanococcus jannaschii, yeast, Methanosarcinaceae species, or E. coli were developed for sitespecific incorporation in E. coli, yeast, or CHO cells.21−27 Third, wild-type synthetases usually do not recognize a nonnatural amino acid. Therefore, researchers need to alter the substrate specificity of the synthetase toward the non-natural amino acid. Schultz and his colleagues developed a powerful screening strategy for generating various synthetase mutants specific for non-natural amino acids.20 Rational design approaches were also utilized to generate synthetase mutants.28 Last, researchers have to generate a new codon or reassign an existing codon for a non-natural amino acid. Among 64 codons, 3 stop codons are not assigned for any natural amino acid. Therefore, one of the three stop codonsin particular, the amber codonhas been widely used to encode non-natural amino acids.12,29−33 Besides stop codons, four-base codons and degenerate codons were utilized as non-natural amino acid incorporation sites.34,35

non-natural amino acid side chain were important for enzyme active site design.

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SITE-SPECIFIC INCORPORATION OF NON-NATURAL AMINO ACIDS TO ENZYMES Site-specific incorporation of non-natural amino acids into proteins has been a powerful tool to engineer proteins including enzymes. Most non-natural amino acids cannot be utilized by living organisms for protein biosynthesis. Therefore, researchers need a special technique involving a non-natural amino acid for protein biosynthesis. Site-specific incorporation allows incorporation of a non-natural amino acid into a specific site of a target protein.20 Here is a general strategy for 6536

DOI: 10.1021/acs.iecr.7b00612 Ind. Eng. Chem. Res. 2017, 56, 6535−6547

Review


Figure 3. Schematic representation of conjugation of RhM-azide to TsFDH-V13pAzF via DBCO-PEG4-DBCO bifunctional linker through SPAAC. Not drawn to scale. Reproduced with permission from ref 55. Copyright 2015 Multidisciplinary Digital Publishing Institute (MDPI).

Figure 4. Characterization of TsFDH-RhM. (a) NAD+-dependent conversion of formate to carbon dioxide by TsFDH-RhM (40 nM), TsFDHV13pAzF (40 nM), TsFDH-WT (40 nM), and RhM (40 nM); (b) photoinduced generation of NADH. The reaction mixture consisting of TEOA (5%), eosin Y (20 μM), NAD+ (0.8 mM), and TsFDH-RhM (40 μM) or free RhM (40 μM) was illuminated by white light at 37 °C. Reproduced with permission from ref 55. Copyright 2015 Multidisciplinary Digital Publishing Institute (MDPI).

Such a site-specific incorporation technique of a non-natural amino acid has been applied to engineer enzymes in a site-specific

manner. In particular, non-natural amino acids with a bioorthogonal functional group have been used to chemically 6537


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Industrial & Engineering Chemistry Research modify enzymes only at a specific site(s) of a target enzyme without compromising the critical properties.

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ENZYME COMPLEX FORMATION USING SITE-SPECIFIC BIOCONJUGATION Enzymes are often conjugated to other molecules for various applications, such as immobilization, delivery, or hybrid formation. In particular, primary amine groups in lysines and sulfhydryl groups in cysteines are usually used as conjugation sites. However, conjugation of enzymes at random sites usually leads to loss in their critical properties due to the blocked active site of enzymes or perturbed folded structure.36−38 To avoid this, conjugation of other molecules to only permissive sites of enzymes is required. Site-specific incorporation of a non-natural amino acid with a reactive functional group can be used to achieve site-specific bioconjugation. In particular, various nonnatural amino acids are available for bioorthogonal chemistries, including click chemistries.39,40 Optimization of CuAAC Conditions for Enzyme Immobilization with the Retained Catalytic Activities. Copper-catalyzed azide−alkyne cycloaddition (CuAAC) is a very popular click chemistry linking an azido and an alkynyl group in the presence of copper catalyst. Since CuAAC does well even under very mild conditions, such as in an aqueous solution at 4 °C, CuAAC has been widely used for biomolecular conjugation applications since its discovery in 2002.41−44 However, surprisingly, applications of CuAAC to engineer enzymes were limited compared to other proteins, because of several issues, including low yield, poor solubility of a ligand, and perturbation of the folded structure by CuAAC components. For example, Candida antarctica lipase B lost a significant portion of catalytic activity upon overnight incubation under CuAAC reaction conditions.45 Upon protein−protein conjugation via CuAAC, Escherichia coli dihydrofolate reductase completely lost catalytic activity due to detrimental effects of the Cu(I) complex.46 Therefore, we reevaluated the effects of CuAAC components on enzyme catalytic activity. For a model system, we chose murine dihydrofolate reductase (mDHFR). The family of DHFRs catalyze conversion of dihydrofolate (DHF) into tetrahydrofolate (THF) using NADPH as a cofactor. To test CuAAC of mDHFR, p-ethynylphenylalanine (pEthF) was site-specifically introduced into the 43ord position of mDHFR to generate mDHFR-43pEthF variant using E. coli expression host cells outfitted with yeast phenylalanyl-tRNA synthetase T415A mutant (yPheRST415A) and yeast phenylalanyl-tRNA amber suppressor mutant (ytRNAPheCUA_UG).47 Next, we evaluated the effects of each CuAAC component on mDHFR activity. TBTA was a popular ligand for CuAAC, though many new ligands have recently been developed. Since TBTA is not water-soluble, TBTA is usually dissolved in dimethyl sulfoxide (DMSO) and then added into the CuAAC reaction mixture. The addition of 30% DMSO led to about 13% activity loss for mDHFR. To overcome this issue, we tested a water-soluble ligand, THPTA. As expected, when THPTA was used in DMSO-free CuAAC reaction solution, the catalytic activity of mDHFR was fully retained. Then, we explored the relative compatibility of reductants in terms of enzyme activity retention. Among three reductants (ascorbate, TCEP, and DTT), ascorbate exhibited the best CuAAC reaction rate with little loss in enzyme activity, whereas DTT and TCEP led to a significant loss in enzyme activity (Figure 1a). Finally, under the optimal CuAAC conditions (1 mM of CuSO4, 1 mM

Figure 5. Schematic showing the click reactions, construction of a multienzyme system, and coupled enzyme reactions. (a) Two bioorthogonal click chemistriesstrain promoted azide−alkyne cycloaddition (SPAAC) and inverse electron-demand Diels−Alder reaction (IEDDA). (b) A strategy to construct a multienzyme reaction system by conjugating two enzymes via consecutive click reactions (SPAAC and IEDDA). NAA, non-natural amino acid containing an azido group; HBL-1, heterobifunctional linker containing dibenzocyclooctyne (DBCO) and tetrazine; HBL-2, heterobifunctional linker containing DBCO and trans-cyclooctene (TCO) groups. (c) The enzymatic D-mannitol synthetic reaction coupled with cofactor regeneration catalyzed by formate dehydrogenase (FDH) and mannitol dehydrogenase (MDH). Reproduced with permission from ref 62. Copyright 2015 Royal Society of Chemistry (RSC).

THPTA, and 2 mM ascorbate), a fluorescence dye was successfully conjugated to a specific site of mDHFR with almost fully retained catalytic activity (Figure 1b). With the optimized CuAAC conditions, biotin was site-specifically conjugated to mDHFR without losing the catalytic activities for enzyme immobilization (Figure 2a). A biotin containing an azide group was conjugated to an ethynyl group of mDHFR43pEthF variant to generate mDHFR-43biotin. The mDHFR43biotin was successfully immobilized on a streptavidin-coated plate, which was confirmed by detection of mDHFR activity, whereas mDHFR-43pEthF without biotin was not well immobilized on the plate supported by the very minimal catalytic activity detected (Figure 2b). The results described in this study47 facilitated researchers’ recognition of the risk of enzyme activity loss upon bioconjugation and needs to 6538


Figure 7. Enzymatic activity assays. Catalytic performance of the FDH−MDH conjugate relative to free enzymes in the multienzyme reaction system for D-mannitol production. The FDH−MDH conjugate (on the basis of 5 nM MDH) or a comparable amount of unconjugated FDH and MDH (5.5 nM dimeric FDH and 5 nM MDH) was subjected to the cofactor-regenerating cascade reaction in the presence of 500 μM NAD+ and 50 mM of formate and D-fructose, respectively. Concentrations of the product, D-mannitol, were measured at 3 and 6 h after the reaction began. Reproduced with permission from ref 62. Copyright 2015 Royal Society of Chemistry (RSC).

optimize reaction conditions to minimize such an activity loss.48−51 Conjugation of an Organometallic Electron Mediator to an Enzyme with the Retained Activities. Although the CuAAC conditions were optimized for enzyme bioconjugation, application of CuAAC is hampered when copper ions reduce

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SPAAC

azide group in p-azido-L-phenylalanine, cyclooctyne group in N6-[(1R,8S,9R)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl-L-lysine or cyclooctyne group in N6-[(2-azidoethoxy)carbonyl]-L-lysine

glutathione S-transferase/ maltose-binding protein

formate dehydrogense/ mannitol dehydrogenase

azide group in p-azido-L-phenylalanine

primary amines in N-terminus and lysines

Michael addition/SPAAC/coupling of primary amine and N-hydroxysuccinimide ester coupling of primary amine and N-hydroxysuccinimide ester in the cross-linker connected to DNA scaffolds SPAAC to the two cross-linkers connected via IEDDA

sulfhydryl group in cysteins, azide group in azidohomoalanine, primary amines in N-terminus and lysines

4-coumarate:coenzyme A ligase/stilbene synthase/ UDP-glucosyltransferase glucose-6-phosphate dehydrogenase/malic dehydrogenase

conjugation chemistry coupling of primary amines and glutaraldehyde

primary amine group in lysines

alcohol dehydrogenase/ lactate dehydrogenase

conjugation sites/functional targets

advantages

site-specific conjugation, good control of enzyme orientation, good control of distance between enzymes site-specific conjugation, no use of crosslinker

good control of distance between enzymes on DNA scaffolds

selective method for coupling more than two enzymes

simple and convenient method

Figure 6. Synthesis and size characterization of the FDH−MDH conjugate. (a) Chemical formulas of AZF and DBCO-derivatized bifunctional linkers. The DBCO group reacted with AZF incorporated into FDH and MDH via SPAAC. The conjugation between FDH and MDH was mediated by IEDDA between tetrazine and TCO. (b) The entire structure of the conjugate focused on the formula for the chemical linker that bridges FDH and MDH. (c) The elution profile of the conjugate by size-exclusion chromatography in comparison to unmodified FDH-WT and MDH-WT. Reproduced with permission from ref 62. Copyright 2015 Royal Society of Chemistry (RSC).

enzymes

Table 1. Strategies of Generating Multienzyme Complexes disadvantages

poor conjugation yield, poor control of distance between enzymes

poor conjugation yield

nonspecific conjugation, partial or complete loss of enzyme activity due to random bioconjugation, random orientation, poor control of enzyme ratio mixed use of non-specific and site-specific conjugation, formation of various sideproducts, needs for complicated purification processes non-specific conjugation, poor control of enzyme orientation

ref

67

62, 66

65

64

63

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Figure 8. Schematic diagrams of orientation variants of the multienzyme nanocomplex and the coupled catalytic reaction. (a) The active sites directed toward each other (face-to-face orientation) and active sites directed away from each other (back-to-back orientation) in the multienzyme complex consisting of dimeric formate dehydrogenase (FDH; orange color) and monomeric mannitol dehydrogenase (MDH; blue color). (b) The enzymatic cascade reactions converting fructose to mannitol by MDH using cofactors supplied from formate conversion into CO2 by FDH. Reproduced with permission from ref 66. Copyright 2016 Nature Publishing Group.

containing an azide group using a bifunctional cross-linker containing dibenzocyclooctyne at both ends via SPAAC (Figure 3). The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity, but it showed some loss in enzymatic activity, probably due to nonspecific interactions between RhM and the TsFDH active site (Figure 4). On the basis of these preliminary results, more studies are required to construct an efficient artificial photosynthesis system utilizing a redox enzyme and an organometallic electron mediator.55 Double Clicking to Couple Multiple Enzymes. In the previous two subsections, conjugation of an enzyme to a solid support and organometallic compound was discussed. However, an enzyme can be coupled to another enzyme for various purposes. In nature, formation of enzyme complexes in cascade enzymatic reactions is often found to enhance overall conversion efficiency via substrate channeling among enzymes.57,58 Similarly, multiple enzymes of our choice can be chemically coupled to facilitate substrate channeling. To enhance control over the coupling site and cross-linking process, several strategies to covalently couple multiple enzymes have been developed.59−61 However, there was still some limitation in choosing coupling sites. In particular, conventional bioconjugation techniques using chemical modification of lysine and cysteine residues often led to large molecular weight conjugates and/or a poorly defined ratio of enzymes in conjugates. In the present study, we described a convenient technique to couple multiple enzymes in a site-specific manner using consecutive click reactions as well as site-specific incorporation of a clickable non-natural amino acid. As a model system, formate dehydrogenase (FDH) and mannitol dehydrogenase (MDH) involved in conversion of D-fructose to D-mannitol via cofactor regeneration were investigated (Figure 5c). A clickable non-natural amino acid, p-azidophenylalanine (AZF), was introduced into a site away from the active site of FDH and MDH with the retained catalytic activity. Then, two different heterobifunctional linkers (Figure 6) were coupled to FDH and MDH via a first click reaction, strain-promoted azide alkyne cycloaddition. Finally, the FDH-linker conjugate was coupled to the MDH-linker conjugate via a second click reaction, the inverse electron demand Diels−Alder reaction (Figure 5a,b).62 The FDH−MDH

Figure 9. Increase in the relative efficiency of the NADH transfer (εrel) for the FF and BB conjugates. The stoichiometry of dimeric FDH and MDH in free enzyme reactions was 1.1:1 which corresponded to the molar composition of FDH-MDH conjugates. Mean ± s.e.m. n = 3. (∗) p < 0.05 (Two-tailed Student’s t test). Reproduced with permission from ref 66. Copyright 2016 Nature Publishing Group.

catalytic activities of a target enzyme. To overcome the issues of copper ion, Bertozzi and her colleagues developed a copper-free click reaction, strain-promoted azide−alkyne cycloaddition (SPAAC).52−54 In SPAAC, instead of a terminal free alkyne for CuAAC, a cyclooctyne group or its derivative is used as a reaction partner with an azide group. Using SPAAC, we explored site-specific conjugation of an organometallic electron mediator to an enzyme with the retained activities (Figure 3), our ultimate goal being the construction of an artificial photosynthesis system.55 In nature, photosynthesis is a series of redox reactions to generate energy-rich chemicals using solar energy. We tested a rhodium-coordinated organometallic electron mediator (RhM) [Rh(bpy) (Cp*)H2O]2+ and formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH)56 as an artificial photosynthesis system (Figure 4). We hypothesized that conjugation of RhM to TsFDH would have several benefits, including enhancement in the overall reaction efficiency and reduction in the amount of expensive RhM. To achieve site-specific conjugation of RhM to TsFDH, TsFDH containing pAzF was linked to an RhM derivative 6540


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Figure 10. Conversion of dihydrofolate (DHF) into tetrahydrofolate (THF) catalyzed by dihydrofolate reductase (DHFR) in the presence of NADPH. Portions of the chemical structure of the murine DHFR inhibitor (methotrexate (MTX)) that differ from DHF are circled. Reproduced with permission from ref 75. Copyright 2013 John Wiley & Sons, Inc.(Wiley).

conjugate exhibited enhanced D-mannitol production compared to free FDH and MDH (Figure 7). These results clearly demonstrated that the site-specific coupling of multiple enzymes was achieved via two compatible click chemistries. The features of the strategy used in this study were compared with those of other strategies (Table 1). Controlling the Active Site Orientation of Multienzyme Complexes. Multistep cascade reactions in nature maximize reaction efficiency by coassembling related enzymes. Such nanostructured enzyme complexes facilitate the processing of intermediates by downstream enzymes, a phenomenon called substrate channeling. Previously, researchers assembled enzymes on a DNA scaffold, demonstrating that the distance between multiple enzymes affects reaction efficiency in the cascade reaction.68−71 However, it remains unknown how the active site orientation controlled at nanoscale can affect multienzyme reaction. Because of technical challenges to control the spatial arrangement of catalytic sites between multiple enzymes, only computational simulation studies proposed the importance of enzyme orientation in multienzyme systems.72,73 Here, we show that controlled alignment of active sites promotes multienzyme reaction efficiency by using genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries. We coupled mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy (Figure 8).66 Coupling of a short linker to a specific residue of each enzyme was achieved by site-specific incorporation of a clickable non-natural amino acid into the specific site, followed by strain-promoted azide−alkyne cycloaddition click chemistry. Then, two enzyme-linker conjugates were coupled via inverse electron demand Diels−Alder click chemistry.66 The study revealed that the multienzyme complex with the active sites directed toward each other exhibits 4-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other (Figure 9). So far, site-specific bioconjugation of an enzyme using a reactive non-natural amino acid was discussed. Therefore, chemical reactivity of non-natural amino acids played an important role. In the next section, properties of non-natural

amino acids other than reactivity were used to modulatethe catalytic properties of enzymes.

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IMPROVEMENT OF ENZYME ACTIVITIES BY SITE-SPECIFIC INCORPORATION Site-specific incorporation of a non-natural amino acid technique allows the introduction of a non-natural amino acid into a specific site in the active site of an enzyme. If a nonnatural amino acid and incorporation site are carefully chosen, the non-natural amino acid incorporation will improve the catalytic properties of the enzyme without compromising other important properties. As a pioneering work, Jackson et al. introduced p-nitrophenylalanine into the active site of E. coli nitroreductase in a site-specific manner, resulting in a 2- to 4-fold increase in catalytic activity compared to that of wildtype enzymes.74 Catalytic properties of enzymes are usually based on interactions between the active site and ligands such as substrates and inhibitors. Since chemical structures of ligands are often so similar, it is very challenging to design the enzyme active site to manipulate ligand interactions with the limited set of 20 natural amino acids. Diversity of size, shape, polarity, and charge in the side chain of non-natural amino acids would facilitate fine-tuning of the enzyme active site. Controlling Enzyme Inhibition Using Non-Natural Amino Acids. One important ligand-enzyme interaction is enzyme inhibition. Enzyme inhibition is critical in the regulation of metabolic pathways and in the design of inhibitorbased pharmaceuticals.75 Furthermore, product inhibition is often a key issue that lowers a conversion yield in industrial enzyme processes. Therefore, controlling enzyme inhibition has been an important topic in enzyme engineering. However, when an inhibitor has a very similar chemical structure with a substrate, inhibitor binding to the active site without compromising substrate binding to the active site is difficult. We explored whether site-specific incorporation of a non-natural amino acid into the enzyme active site can be used to reduce enzyme inhibitor binding without reducing substrate binding. As a model system, we chose murine dihydrofolate reductase (mDHFR), substrate dihydrofolate (DHF), and inhibitor methotrexate (MTX). The family of DHFRs catalyzes the DHF conversion into tetrahydrofolate (THF) using NADPH as a cofactor (Figure 10). MTX is a very effective competitive 6541


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Figure 11. Multiple sequence alignment of mammalian DHFRs and the structural models of DHFR:ligand complexes. (a) The amino acid sequences obtained from five different mammals (mouse, rat, human, monkey, and cattle) were aligned: (:) conserved residues; (.) semiconserved residues; ( ∗) strictly conserved residues). (b) Chemical structures of phenylalanine (Phe), p-bromophenylalanine (pBrF), and L-2-naphthylalanine (2Nal). (c) The crystal structure of hDHFRWT:inhibitor (PDB ID: 1U72) (top left); the structural model of mDHFRpBrF31:inhibitor (top middle) and mDHFR2Nal31:inhibitor complex (top right). The structural models of mDHFRWT:substrate (wild-type human DHFR:folate complex; PDB ID: 2W3M) (bottom left); mDHFRpBrF31:substrate (bottom middle) and mDHFR2Nal31:substrate (bottom right). Inhibitor (orange); substrate (bright orange); carbon atom in side chain (magenta); bromine atom in side chain (light pink). Reproduced with permission from ref 75. Copyright 2013 John Wiley & Sons, Inc.(Wiley).

Among amino acids that directly contact substrate DHF and inhibitor MTX, phenylalanine at position 31 (F31) was chosen as a site for non-natural amino acid incorporation. Then, phenylalanine analogues to replace F31 were chosen using a structural model of mDHFR and rotamer libaries of phenylalanine analogues. Two structural models of mDHFRs complexed with DHFR or MTX were constructed based on the crystal structures of human DHFRs highly homologous to mDHFR. Rotamer structural files of Phe analogues were obtained from the SwissSide chain database.76 Without energy minimization, the structural models complexed with either DHF or MTX were constructed to visualize the extent of ligand steric clash with the active site residues in order to estimate the

Table 2. MTX Dissociation Constants and DHF Kinetic Parameters of mDHFR Variants mDHFR variant

WT

pBrF

2NaI

KD (μM) KM (μM) kcat (s−1) KD/KM

8.9 ± 3.8 6.5 ± 1.0 0.32 ± 0.01 1.4 ± 0.6

18.4 ± 6.4 3.4 ± 0.6 0.42 ± 0.02 5.4 ± 2.1

37.9 ± 8.8 4.8 ± 1.6 0.085 ± 0.007 7.9 ± 3.2

Values means ± standard errors. Reproduced with permission from ref 75. Copyright 2013 John Wiley & Sons, Inc.

inhibitor to mammalian dihydrofolate reductases (DHFRs), because it has a very similar structure to that of substrate DHF (Figure 10). 6542


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Figure 12. Reactions catalyzed by DHFR and crystal structure of DHFR. (A) Conversion of folate (FOL) into dihydrofolate (DHF) and tetrahydrofolate (THF) catalyzed by dihydrofolate reductase (DHFR) in the presence of NADPH. (B) Crystal structure of hDHFR complexed with substrate folate (PDB ID: 2W3M). Folate (red); six residues (R28, E30, F31, Y33, F34, and Q35) close to folate (green). Reproduced with permission from ref 16. Copyright 2015 Elsevier.

stability of mDHFR-ligand complex (Figure 11). In the structural models, replacement of F31 with two phenylalanine analogues (p-bromo-phenylalanine [pBrF] and 2-naphthylalanine [2Nal]) leads to steric clash with MTX, whereas it does not cause steric clash with DHF. Next, two mDHFR variants containing either pBrF (mDHFRpBrF31) or 2Nal (mDHFR2Nal31) at position 31 were prepared using the triple autotrophic AFWK E. coli expression host outfitted with yeast phenylalanyl-tRNA synthetase (yPheRS) mutant and yeast phenylalanyl-tRNA amber suppressor mutant (ytRNACUA_UG). Then, we discovered that mDHFRpBrF31 and mDHFR2Nal31 possess a binding affinity toward the substrate DHF greater than the inhibitor MTX by 4.0- and 5.8-fold, respectively. Such an enhanced selectivity is mainly attributed to a reduced inhibitor binding affinity by 2.1- and 4.3-fold, respectively (Table 2). Although this work demonstrated the concept of enzyme inhibition control using the active site modification with a non-natural amino acid, this approach was not general enough to apply systematically to other enzymes. Therefore, we further explored whether a rational design of the enzyme active site using a non-natural amino acid could be achieved. Manipulating Substrate Specificity of an Enzyme Using Non-Natural Amino Acid Incorporation. Another important ligand−enzyme interaction is substrate−enzyme interaction. When an enzyme can utilize multiple substrates, manipulating substrate specificity of an enzyme is often required. For a rational design, we investigated the computational approach to design the enzyme active site for the enhanced catalytic efficiency using site-specific incorporation of a nonnatural amino acid. Kuhlman and Baker developed a powerful computational protein-design program, RosettaDesign.77,78

RosettaDesign can be used to predict the energy function of protein folding, and hence the conformational stability, of a protein. Furthermore, Baker and his colleagues developed a computational ligand-docking program, RosettaLigand.79,80 RosettaLigand takes the structures of a ligand and its receptor protein and then calculates the energy function to identify a conformation and relative orientation of the receptor and ligand. Originally, both RosettaDesign and RosettaLigand used rotamer libraries of only natural amino acids. Kuhlman and his colleagues expanded the rotamer libraries to include various non-natural amino acids and make those non-natural amino acids available for RosettaDesign and RosettaLigand.81 Using RosettaDesign and RosettaLigand as well as non-natural amino acid rotamer libraries, we investigated whether site-specific incorporation of a non-natural amino acid into the enzyme active site can manipulate the substrate specificity. For a model system, we chose mDHFR and its two substrates, DHF and folate (FOL). DHFRs catalyze the conversion of a relatively poor substrate FOL into a relatively good substrate DHF followed by the conversion of DHF into tetrahydrofolate using NADPH as a cofactor (Figure 12a). Although the chemical structure of FOL differs slightly from DHF, the activity of human DHFR toward FOL is about 30 times lower than that toward DHF.82 Such a poor DHFR activity toward FOL was attributed to a weaker binding affinity to DHFR compared to DHF. We hypothesized that the site-specific incorporation of a non-natural amino acid into mDHFR can be used to redesign the active site for the altered substrate specificity between FOL and DHF. RosettaDesign and RosettaLigand were used to predict the mDHFR variant containing 2Nal with the altered substrate specificity toward FOL over DHF. From the crystal structure of 6543


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Industrial & Engineering Chemistry Research Table 3. Scoring Summary of mDHFR Variantsa 2NaI incorporation site

FOL docking score (kcal/mol)

DHF docking score (kcal/mol)

docking score difference (FOL − DHF)c (kcal/mol)

conformational stability score (kcal/mol)

noneb R28 E30 F31 Y33 F34 Q35

−504 −542 −509 −541 −520 −529 −526

−495 −537 −499 −477 −510 −487 −523

−9 −5 −10 −64 −10 −42 −3

−329 −323 N.D.d −325 −288 −246 −303

a

Reproduced with permission from ref 16. Copyright 2015 Elsevier. bmDHFRWT. cThe docking score for FOL subtracted by the docking score for DHF. dThe score was not obtained due to an calculation error.

Table 4. Kinetic Parametersa of mDHFRWT and mDHFR2Nal31 b substrate c

DHF

FOL

kinetic parameters KM (μM) kcat (s−1) kcat/KM (s−1 μM−1) Km (μM) kcat (s−1)

mDHFRWT 6.5 ± 1.0 0.32 ± 0.01 4.9 × 10−2 ± 7.7 × 10−3 22.5 ± 7.5 7.9 × 10−3 ± 7.3 × 10−4 3.5 × 10−4 ± 1.2 × 10−4 3.5

kcat/KM (s−1 μM−1) Relative values KM for FOL/KM for DHF 7.1 × 10−3 kcat/KM ratiod kcat/KM 1 ratio(rel)e

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CONCLUSIONS AND OUTLOOK

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AUTHOR INFORMATION

Since its debut in 2001, site-specific incorporation of a nonnatural amino acid in vivo has been a powerful tool for engineering proteins, including enzymes. Besides expansion of the number of non-natural amino acids available for site-specific incorporation, this approach has been extended to yeast, mammalian cells, worms, and fish.83−86 In other words, we can obtain a recombinant protein containing a non-natural amino acid at a specific site using various types of expression hosts. Furthermore, this technique is becoming a general tool used by numerous researchers worldwide. Great efforts have been made to increase the accessibility of this technique to researchers. Many plasmids encoding an orthogonal pair of tRNA/ synthetase specific to a non-natural amino acid are available from developers, as well as Addgene, though some non-natural amino acids are not yet commercially available. On the basis of the popularity of the site-specific incorporation technique as a protein engineering tool, we can easily imagine its diverse applications in engineering enzymes, industrially important catalytic proteins. In this review, we summarized recent studies on the bioconjugation of enzymes via click chemistries to other molecules, such as fluorescent dyes, biotin, and other enzymes. Since such a site-specific bioconjugation of an enzyme allows retention of catalytic properties, immobilization of enzymes would be a very promising application. Although the relatively high cost of non-natural amino acid and click reagents is still a hurdle for industrial applications, biomedical or biopharmaceutical applications seem feasible. We also briefly mentioned enzyme active site design/engineering using non-natural amino acids. Because of technical difficulties, this area is being developed slowly compared to bioconjugation of enzymes. However, thanks to advances in computational protein design, we expect to see more examples in the design of novel enzymes containing a non-natural amino acid in the active site in the near future.

mDHFR2Nal31 4.8 ± 1.6 0.085 ± 0.007 1.8 × 10−2 ± 6.1 × 10−3 9.5 ± 2.8 9.1 × 10−3 ± 5.5 × 10−4 9.6 × 10−4 ± 2.9 × 10-4 2.0 5.4 × 10−2 7.6

Values ± regression standard error. bReproduced with permission from ref 16. Copyright 2015 Elsevier. cTaken from ref 75. d (kcat/KM for FOL)/(kcat/KM for DHF). eRelative to kcat/KM ratio of mDHFRWT. a

the DHFR:FOL complex, six residues (R28, E30, F31, Y33, F34, and Q35) within 5 Å of substrate FOL were selected for 2Nal incorporation (Figure 12b). Then, structural models of mDHFR variants containing 2Nal at one of the six residues selected were constructed. RosettaDesign was used to compare the conformational stabilities of the six mDHFR variants as well as wild-type mDHFR (mDHFRWT). RosettaLigand was used to compare the docking scores (relative binding affinities) of mDHFR variants containing 2Nal toward DHF and FOL. The mDHFR variant containing 2Nal at position 31 (mDHFR2Nal31) exhibited the maximum docking score difference between FOL and DHF, indicating the enhanced binding affinity toward FOL compared to DHF (Table 3). These results indicate that the mDHFR2Nal31 variant was the best candidate in terms of potential enhanced substrate specificity toward FOL. Therefore, purified mDHFRWT and mDHFR2Nal31 variant were experimentally prepared and subjected to kinetic assays. The kinetic assays revealed that the mDHFR2Nal31 actually has enhanced binding affinity toward FOL over DHF, and furthermore, it exhibited kcat/KM ratio (kcat/KM for FOL over kcat/KM for DHF) 7.6 times greater than mDHFRWT (Table 4). This approach was a rational enzyme design based on the computationalprotein design tools RosettaDesign and RosettaLigand. Considering the general nature of the approach and techniques, this approach has great potential to alter the substrate specificity of other enzymes.

Corresponding Author

*E-mail: [email protected]. Tel.: +82 62-715-2312. Fax: +82 62-715-2304. ORCID

Inchan Kwon: 0000-0003-0806-4116 Notes

The authors declare no competing financial interest. 6544


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Industrial & Engineering Chemistry Research Biographies

(2) Estell, D. A.; Graycar, T. P.; Wells, J. A. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 1985, 260 (11), 6518−21. (3) Neuner, P.; Cortese, R.; Monaci, P. Codon-based mutagenesis using dimer-phosphoramidites. Nucleic Acids Res. 1998, 26 (5), 1223− 7. (4) Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J. Am. Chem. Soc. 2013, 135, 12480. (5) Francis, J. C.; Hansche, P. E. Directed evolution of metabolic pathways in microbial populations. I. Modification of the acid phosphatase pH optimum in S. cerevisiae. Genetics 1972, 70 (1), 59−73. (6) Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nat. Chem. Biol. 2009, 5 (8), 567−73. (7) Hu, Q.; Xu, Y.; Nie, Y. Highly enantioselective reduction of 2hydroxy-1-phenylethanone to enantiopure (R)-phenyl-1,2-ethanediol using Saccharomyces cerevisiae of remarkable reaction stability. Bioresour. Technol. 2010, 101 (22), 8502−8. (8) Rothlisberger, D.; Khersonsky, O.; Wollacott, A. M.; Jiang, L.; DeChancie, J.; Betker, J.; Gallaher, J. L.; Althoff, E. A.; Zanghellini, A.; Dym, O.; Albeck, S.; Houk, K. N.; Tawfik, D. S.; Baker, D. Kemp elimination catalysts by computational enzyme design. Nature 2008, 453 (7192), 190−5. (9) Arnold, F. H. Combinatorial and computational challenges for biocatalyst design. Nature 2001, 409 (6817), 253−7. (10) Jiang, L.; Althoff, E. A.; Clemente, F. R.; Doyle, L.; Rothlisberger, D.; Zanghellini, A.; Gallaher, J. L.; Betker, J. L.; Tanaka, F.; Barbas, C. F., 3rd; Hilvert, D.; Houk, K. N.; Stoddard, B. L.; Baker, D. De novo computational design of retro-aldol enzymes. Science 2008, 319 (5868), 1387−91. (11) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 2001, 292, 498−500. (12) Mendel, D.; Cornish, V. W.; Schultz, P. G. Probing Protein Structure and Function with an Expanded Genetic Code Annu. ReV. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 435−462. (13) Budisa, N. Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew. Chem., Int. Ed. 2004, 43 (47), 6426−6463. (14) Link, A. J.; Tirrell, D. A. Reassignment of sense codons in vivo. Methods 2005, 36, 291−298. (15) Budisa, N.; Wenger, W.; Wiltschi, B. Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastoris. Mol. BioSyst. 2010, 6 (9), 1630−9. (16) Zheng, S.; Lim, S. I.; Kwon, I. Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids. Biochem. Eng. J. 2015, 99, 85−92. (17) Ayyadurai, N.; Deepankumar, K.; Prabhu, N. S.; Lee, S.; Yun, H. A facile and efficient method for the incorporation of multiple unnatural amino acids into a single protein. Chem. Commun. 2011, 47 (12), 3430−3432. (18) Ayyadurai, N.; Prabhu, N. S.; Deepankumar, K.; Jang, Y. J.; Chitrapriya, N.; Song, E.; Lee, N.; Kim, S. K.; Kim, B.-G.; Soundrarajan, N.; Lee, S.; Cha, H. J.; Budisa, N.; Yun, H. Bioconjugation of l-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide. Bioconjugate Chem. 2011, 22 (4), 551−555. (19) Lim, S. I.; Kwon, I. Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids. Crit. Rev. Biotechnol. 2016, 1−13. (20) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 2001, 292, 498−500. (21) Kwon, I.; Wang, P.; Tirrell, D. A. Design of a bacterial host for site-specific incorporation of p-bromophenylalanine into recombinant proteins. J. Am. Chem. Soc. 2006, 128, 11778−11783. (22) Plass, T.; Milles, S.; Koehler, C.; Schultz, C.; Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem., Int. Ed. 2011, 50, 3878−3881. (23) Seitchik, J. L.; Peeler, J. C.; Taylor, M. T.; Blackman, M. L.; Rhoads, T. W.; Cooley, R. B.; Refakis, C.; Fox, J. M.; Mehl, R. A. Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific

Inchan Kwon received his BS (1994) and MS (1996) in Chemical Engineering from Seoul National University (South Korea). He obtained his Ph.D. at Caltech under the direction of Professor David A. Tirrell, and completed his postdoctoral training in Chemical Engineering (2008) at the University of California at Berkley. After his career as an Assistant Professor at Department of Chemical Engineering at the University of Virginia starting in 2008, he joined School of Materials Science and Engineering at GIST (South Korea) as an Associate Professor. His main research interest lies in engineering and bioconjugation of therapeutic or industrial enzymes.

Byungseop Yang was born in Gwangju, South Korea, in 1989. He studied chemistry and received a bachelor’s degree in 2014 from Chonnam National University, Gwangju, South Korea. Afterwards, he received an MS degree in School of Materials Science and Engineering at Gwangju Institute of Science and Technology (GIST), Gwangju, South Korea, under the direction of Professor Inchan Kwon in 2016. He continued his Ph.D. degree under the direction of Professor Inchan Kwon. His research involves serum half-life extension of therapeutic enzymes in vivo using site-specific bioconjugation.

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ACKNOWLEDGMENTS This invited contribution is part of the I&EC Research special issue for the 2017 Class of Influential Researchers. The authors acknowledge financial support from Korea C1 Gas Refinery Program (Grant No. 2015M3D3A1A01064923) funded by the Ministry of Science, ICT & Future Planning and the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. 2014H1C1A1067014).

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Bioconjugation and Active Site Design of Enzymes Using Non-natural

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