Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

doi:10.21203/rs.3.rs-468406/v2

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Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

https://doi.org/10.21203/rs.3.rs-468406/v2

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Here, incorporation of secondary amine by genetic code expansion was used to expand the potential protein templates for artificial enzyme design. Pyrrolysine analogue containing a D-proline could be stably incorporated into proteins, including the multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Both modified scaffolds were catalytically active, mediating transfer hydrogenation with a relaxed substrate scope. The protein templates played a distinctive role in that, while the LmrR variants were confined to the biomimetic BNAH as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for reactions. Due to the cofactor compatibility, the DHFR secondary amine catalysis could also be coupled to an enzymatic recycling scheme. This work has illustrated the unique advantages of using proteins as hosts, and thus the presented concept is expected to find uses in enabling tailored secondary amine catalysis.

Chemical Biology

Artificial Enzyme

Genetic Code Expansion

Secondary Amine Organocatalysis

Iminium Catalysis

Protein Engineering

The use of secondary amines as catalytic motifs has tremendous potentials in artificial enzyme design. In nature, primary amines including the amino groups of lysine residues and protein N-termini are widely used in enzyme catalysis, serving as an acid, a base or nucleophile.^1-2 In contrast, the use of secondary amines in enzyme catalysis is significantly rarer and their scope has not been fully explored.^3-7 However, while the basicity of cyclic secondary amines are similar to their primary counterparts, they are significantly more nucleophilic.^8-10 Additionally, when a secondary amine reacts with a carbonyl substrate, the resulting iminium ion intermediate does not contain a proton on the nitrogen atom; this can prompt reactions with a latent nucleophile or a base for enamine formation, driving the catalytic cycles forward.^11-13 Indeed, proline and its derivatives (e.g., prolyl-RNA) have been proposed to play roles in the prebiotic world catalyzing the formation of crucial building blocks such as carbohydrates and nucleotides.^14-16 Given the reactivity of secondary amines, their incorporation into protein scaffolds can generate catalytically active entities which have strong potentials to be transformed into highly active and selective artificial enzymes.

Currently, only two types of templates have been used to generate protein-based secondary amines for catalysis. 4-Oxalocrotonate tautomerase (4-OT) contains a N-terminal proline residue within its cavity, and it can be used to mediate iminium or enamine catalysis.^17-20 In the second approach, pyrrolidines covalently linked to biotin were introduced to streptavidin (Sav) affording a protein-hosted secondary amine catalytic system.^21-23 These protein catalysts could catalyze various types of reactions, including conjugate addition,^19-21 aldol condensation,^{18, 22} transfer hydrogenation²³ and epoxidation.²⁴ However, since there are only a few proteins that contain a N-terminal proline within their cavity^{3, 6} and also only a few that bind biotin with significant affinity,^25-26 the choice of protein templates for hosting secondary amine is limited. Indeed, the full benefit of using protein as host has not been revealed, and a generic approach that facilitates the incorporation of secondary amine is needed.

Genetic code expansion has the advantage of fewer restrictions on the choice of protein scaffolds and the site therein.^27-31 Previously, an aniline motif has been added to the multidrug binding protein LmrR by incorporating unnatural amino acid p-azidophenylalanine followed by chemical reduction.^27-30 Whilst being less nucleophilic than pyrrolidine,^{8, 10} the aniline in LmrR was able to catalyze various carbon-heteroatom and carbon-carbon ligation reactions.^27-30 Another example is the introduction of a N-methyl histidine residue into the computationally designed protein BH32;³¹ subsequent laboratory evolution afforded a catalyst capable of hydrolyzing fluorescein esters through formation of a covalent intermediate. Previously, secondary amines have been incorporated into protein scaffolds (e.g., β-galactosidase) as pyrrolysine analogues, in which proline and its derivatives were attached to lysine through isopeptide bonds.³² However, their applications in artificial enzyme design have yet to be explored. Furthermore, stability of these proline derivatives is unknown, as they can be proteolytically hydrolyzed during recombinant preparation. Nevertheless, pyrrolysine analog incorporation is worth exploring because it can be an effective approach to screen a broad range of protein templates through introduction of stable, catalytically active secondary amines.

In this work, through genetic code expansion, unnatural amino acids 1-3 bearing a cyclic secondary amine were site-specifically introduced into different scaffolds, including the super folder green fluorescent protein (sfGFP), multidrug-binding protein LmrR and nucleotide-binding dihydrofolate reductase (DHFR) (Fig. 1A).³³ Hydrolysis of the isopeptide bond was observed for proteins incorporated with 2 (L-proline) but not detected for proteins incorporated with 1 (D-proline) or 3 (L-thioproline). Incorporation of 1 resulted in catalytically active entities, and the effect of the protein template has been illustrated in the reactions catalyzed by the corresponding LmrR and DHFR variants. While both systems could catalyze the model reduction of various α,β-unsaturated aldehydes (Fig. 1B & C), the LmrR-hosted catalytic system could only use the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as a source of hydride. In contrast, a DHFR variant was found to recruit the pro-R hydride from the cofactor NADPH for transfer hydrogenation. Further investigations revealed that the hydride delivery in the DHFR system is not rate-limiting, and the catalysis can be coupled to an enzymatic NADPH regeneration scheme. This work has proved the concept that genetic code expansion is a viable tool to examine different protein templates for designed secondary amine catalysis.

Unnatural amino acids containing a secondary amine were tested for incorporation (Fig. 1A). Amino acids 1, 2, and 3 are derived from D-proline, L-proline and L-thioproline (L-thiazolidine-4-carboxylic acid), respectively (see SI for their synthesis). Based on previous reports, 1 and 3 are substrates of the wild-type Methanosarcina bakeri pyrrolysyl-tRNA synthetase (MbPylRS) and its engineered variant ThzKRS, respectively.^{32, 34} Due to the structural similarity between 2 and 3, we envisaged that 2 could be a substrate of ThzKRS. Incorporation of unnatural amino acids was tested using a version of sfGFP whose gene bears a TAG codon in the middle (i.e., 150^th amino acid residue, Asn150TAG), and formation of the full-length protein implied successful incorporation of the unnatural amino acid as indicated by SDS-PAGE analysis (Fig. S1 and Table S1).³⁵ However, since the corresponding unnatural amino acid residue is located at a solvent exposed position, rather than the interior of the protein, their stability and catalytic activity were not further examined.

For catalyst design, the unnatural amino acids were incorporated into the multidrug binding protein LmrR,³⁶ a scaffold known to be catalytically competent after modification of its hydrophobic pocket including the insertion of an unnatural amino acid.^37-40 Hence, we selected four previously reported residues, Val15, Asn19, Met89 and Phe93 (Fig. 1B), located in the hydrophobic pocket for substitution with 1, 2 or 3. The 12 LmrR variants were successfully produced by Escherichia coli as revealed by SDS-PAGE analysis (Fig. S2). The dimeric nature of these variants was confirmed by size exclusion chromatography, with the exception of LmrR-Met89-2 whose oligomeric nature appears to be hampered by the modification (see SI, Pg S10-12). However, liquid chromatography-mass spectrometry (LC-MS) investigation indicated that variants containing amino acid 2 were unstable and up to ~35% hydrolysis of the L-prolyl group was observed at position 89 (Fig. 2). Direct incorporation of lysine was ruled out as the full-length protein could not be obtained without supplementing 2 in the growth cultures. Hence, this observation was attributed to the hydrolysis of the L-prolyl group during protein production, as E. coli contains enzymes such as proline iminopeptidase which can cleave the N-terminal proline in polypeptides (e.g., UniProt: A0A6N7NN15_ECOLX). In contrast, truncation was not observed in proteins incorporated with either D-proline 1 or L-thioproline 3, and hence they were concluded non-hydrolysable.

We chose the conversion of cinnamaldehyde 4 to its reduced counterpart 5 as the model reaction (Fig. 3) because the streptavidin (Sav)-hosted pyrrolidine was shown to be able to catalyze this reaction,²³ and hence direct comparison among the protein-based catalytic systems can be made. BNAH, unlike other biomimetics such as Hantzsch ester, is soluble in aqueous buffers and thus was tested as the hydride donor.²³ Conversion of the reaction was estimated using a gas chromatography-mass spectrometry (GC-MS) setup as previously described (see SI).⁴¹ Though containing seven lysine residues and a free N-terminal amino group, the wild-type LmrR could only catalyze less than 3% of conversion implying minimal activity (Fig. 3A and Table S2). Substitution of Met89 with 1, 2 or 3 also showed marginal improvement (~5% conversion). For all the variants incorporated with 2 of which truncation was detected, low conversion (<15%) was observed. Substitution of thioproline 3 at position 19 and D-proline 1 at position 15 resulted in a detectable amount of product conversion (> 20%). Nevertheless, the replacement of Phe93 with 1 (LmrR-Phe93-1) was most promising giving a yield 19-fold higher than that of the wild-type enzyme (58% vs. 3%). LmrR-Phe93-2 differs by one stereocenter (D- vs L-proline) and, while a majority contain the secondary amine (~80%, see Fig. 2), it could hardly catalyze the model reaction (<2%). In contrast, LmrR-Phe93-3, the non-hydrolysable thiazolidine equivalent of LmrR-Phe93-2, was able to mediate a lower but detectable amount of conversion (12%). Accordingly, several factors, including the chirality, stability and electron density of the catalyst, appear to play roles in the extent of conversion. NADPH was tested as the hydride donor for catalysis by LmrR-Phe93-1, but no conversion was detected. Transfer hydrogenation with NADPH by unnatural amino acid 1 was also not detectable (Fig. 3B). These observations align with the literatures. The secondary amines hosted by Sav could not recruit NADH for reaction in aqueous buffer.²³ Similarly, NADH was found to be a poor hydride donor for small organocatalytic secondary amines.^42-43 Indeed, while examples of metal catalysts and artificial metalloenzymes using NAD(P)H or NAD(P)⁺ for reactions have been reported,^44-48 the equivalents in organocatalytic system are much rarer.

Seeing if NADPH can be used as a hydride source by a switch of template, the E.coli dihydrofolate reductase (DHFR) was tested. DHFR catalyzes the step of hydride transfer from the C4 pro-R hydride of NADPH to C6 of dihydrofolate.^49-51 Containing a Rossmann fold, DHFR is composed of a multi-stranded β sheet packed by two α helices on each side and binds nucleotide-containing cofactor NADPH with an association rate up to 10⁵ M^-1·s^-1 (Fig. 1C).^50-51 Additionally, it contains a mobile M20 loop (residue 9-23) which closes the active site upon binding NADPH,

thereby generating a shielded environment surrounding the nicotinamide motif.^52-53 These features present DHFR as a plausible scaffold for transfer hydrogenation by NADPH. Hence, Ala7, Phe31 and Ser49 that are in proximity to the nicotinamide motif were individually replaced with 1, the non-hydrolysable unnatural amino acid that gave the highest conversion in the previously examined system (LmrR). The resulting DHFR secondary amines were verified by SDS-PAGE (Fig. S3) and mass spectrometry (Fig. S4). Similarly, hydrolysis of the D-prolyl group was not detected.

Activity tests revealed that DHFR variants incorporated with 1 could facilitate NADPH-dependent transfer hydrogenation (Fig. 3B). For the wild-type DHFR which contains six lysine residues and a N-terminal amino group, the reaction product 5 was undetectable by GC-MS analysis. Replacement of Ser49 with 1 led to considerable protein precipitation with no detectable products. Contrarily, replacement of Ala7 with 1 could effectively catalyze the reaction as evident by significant product conversion (Fig. 3B and Table S3). Though BNAH can be used as the hydride source, the extent of product conversion was noticeably higher when utilizing NADPH. With 0.1 equivalent (10 mol%) of DHFR-Ala7-1, 90% of starting material 4 was converted into product 5 in aqueous PBS buffer at pH 7.0 using 2.0 equivalents of NADPH after 18 hours, while ~34% conversion with BNAH was achieved under the same condition (Table S4).

Similar to many other secondary amine organocatalysts,⁹ formation of iminium ion is critical towards carbonyl substrate activation, and thus its transient existence during the catalysis by LmrR-Phe93-1 and DHFR-Ala7-1 was probed. The two variants were incubated with the starting material 4, treated with NaCNBH₃ and subjected to high resolution mass spectrometric analysis, similar to previously described (see SI).²⁸ The LmrR-Phe93-1 sample predominantly yielded one protein species with an observed mass matching the calculated molecular weight of the reduced covalent protein-substrate intermediate (Fig. S5A). Similarly, a substantial portion of the DHFR-Ala7-1 variant also yielded the reduced covalent intermediate under the same condition. However, there was a noticeable amount of unmodified DHFR-Ala7-1, suggesting this variant is less reactive and the secondary amine in 1 is less accessible (see below for kinetic measurement). Interestingly, hydrolysis of the D-prolyl group was also observed upon treatment with NaCNBH₃(Fig. S5B). Treatment of these protein species with chymotrypsin revealed digested peptides, whose elemental composition identified by liquid chromatography mass spectrometry (LC-MS) corresponds to the trapped intermediate (Fig. S6 and S7). In contrast, the wild-type LmrR and DHFR that cannot catalyze the transfer hydrogenation did not show any evidence of the iminium intermediate formation. Accordingly, these results support a LUMO-lowering reaction mechanism,⁹ in which the secondary amine activates the starting material through iminium ion formation for transfer hydrogenation (Fig. 4).

Contrary to the previous work which used a LmrR-hosted aniline for catalysis,²⁸ no lysine modification was detected in our protein scaffolds after NaCNBH₃ treatment. The chemoselectivity observed here implies the significantly higher reactivity of the secondary amine which forms iminium intermediate with relatively inert carbonyl substrate under aqueous conditions. Furthermore, in LmrR the aniline organocatalysis was found to be optimal at position 15,²⁸ whereas we obtained higher conversions with the secondary amine organocatalyst at position 93, indicating that activity test at different positions is essential during protein-based catalyst design, an experiment that can be readily achieved by genetic code expansion.

The k_cat constants were estimated to be highest for the previously reported streptavidin-hosted secondary amine system (>20 fold, ~0.01275 s^-1),²³ followed by LmrR (~2 fold) and eventually DHFR which recruits NADPH as the hydride donor instead of BNAH (Table S5 & S6, Fig. S8 and Pg S17-18 for assessment). These differences can be explained by the locations of the catalytic centers. The Sav-hosted secondary amine is most solvent exposed²¹ and thus it can readily react with the carbonyl substrate contributing to a large k_cat constant. In contrast, in both LmrR-Phe93-1 and DHFR-Ala7-1 the secondary amines are less accessible locating within the protein cavities and lower k_cat constants were observed. However, the estimated Michaelis constant (K_M) value for the corresponding hydride donor was found to be lowest for DHFR (94 μM), resulting in an enhancement in the bimolecular rate constant that is approximately 6-fold higher than that of the LmrR system. Accordingly, though the secondary amine may be less accessible in the DHFR variant, its tight binding nature to NADPH facilitates the step of hydride transfer, hence the significant conversion observed (Fig. 3 and 5, also see below). On the other hand, the K_M for cinnamaldehyde could not be accurately determined due to precipitation (in aqueous buffer containing 10% methanol). Lastly, the estimated bimolecular rate constants for all three systems were considerably lower than those for natural enzymes that recruit BNAH or NADPH for reactions.^54-55 Nevertheless, this work proves the concept that incorporation of secondary amine by genetic code expansion facilitates reactions with the designated nucleophile (NADPH vs. BNAH), and performance of these first-generation catalysts can be readily enhanced through established techniques.^{27, 56-57}

When [4R-²H]-NADPH (NADPD) was used in the catalysis by DHFR-Ala7-1, only one product isotopologue with the deuteride located at the C_β position was identified by GC-MS (Fig. S9). This observation agrees with previous crystal and biochemical analysis which illustrated that the wild-type DHFR catalyzes the transfer of the C4 pro-R hydride of NADPH.^58-59 Furthermore, this protein catalyst is regioselective favoring 1,4- over 1,2-addition, the latter was observed in the Sav-hosted secondary amine catalytic system as a side reaction.²² Given its selectivity, primary kinetic isotope effect (KIE) of hydride transfer was assessed. The turnover rate constants under saturating conditions between the reactions that use NADPH and NADPD yielded a kinetic isotope effect (KIE) of 1.1 ± 0.2 (Fig. S10), implying that the step of hydride transfer is not rate-limiting. Other chemical step(s) such as iminium ion formation can be rate-limiting.⁶⁰ Alternatively, the physical step of releasing the oxidized cofactor from the enzyme can dictate the rate of catalytic turnover, as observed in the natural reaction catalyzed by the wild-type enzyme.^{50, 53} Subsequently, the stereoselectivity of hydride transfer was investigated by use of the prochiral substrate β-methylated cinnamaldehyde. Though product formation was observed, its low conversion (24%) has prevented us from accurately accessing the enantioselectivity through chiral LC analysis (Fig. 5). Hence, for future work both the reactivity and stereoselectivity of reactions with β-alkylated cinnamaldehydes will be optimized through established protein engineering techniques such as directed evolution.^61-64

Substrate scope of LmrR-Phe93-1 and DHFR-Ala7-1 were investigated. Aromatic and ketone analogues of cinnamaldehyde were evaluated as alternative substrates. Both systems were able to accept the aldehydes as substrates. Although different hydride donors were used, product conversions by DHFR-Ala7-1 were generally higher than those by LmrR-Phe93-1 with respect to per mol% of catalyst (Fig. 5, Table S7 and S8). However, the ketone substrate showed no turnover in either system.

Introducing catalytic motifs by genetic code expansion has allowed us to explore different protein scaffolds and hence discover a NADPH-dependent secondary amine catalyst. Although NADPH is structurally complex, catalysis by DHFR-Ala7-1 could be coupled with an enzymatic cofactor regeneration scheme (Fig. 6A). Importantly, this experiment proves the concept that secondary amine organocatalysis, amongst other chemical catalytic systems,⁶⁵ can be driven by a well-established biocatalysis tool with the potential to be integrated into live cells. The conversion of starting material 4 to product 5 by DHFR-Ala7-1 was performed in the presence of the reaction by glucose 6-phosphate dehydrogenase (G6PDH), a commercially available enzyme that oxidizes glucose 6-phosphate whilst reducing NADP⁺ to regenerate NADPH.⁶⁶ The product conversion could reach 90% (10 μM NADPH) when the coupling reaction was included, whereas under the same reaction conditions the conversion was barely detectable (<1%) in the absence of G6PDH (Fig. 6B). A significant drop in conversion was observed when [NADPH] was less than 1 μM; a plausible explanation is that the DHFR variant becomes destabilized without cofactor binding. Nevertheless, the turnover number for NADPH (ratio of product formed to NADPH used) can reach as high as 10⁴ (Fig. 6C).

Secondary amines are more nucleophilic than their primary counterparts, and their incorporations have converted poorly active, lysine-containing proteins into catalytically active entities. Previous approaches are limited to only a handful of protein templates.^17-23 Here, this limitation on protein templates has been majorly alleviated by use of genetic code expansion, through which secondary amines were introduced to different scaffolds (sfGFP, LmrR and DHFR) at the position of choice. As a proof of concept, a secondary amine catalyst with unique cofactor dependence has been promptly established, indicating the benefit of using proteins as hosts. Provided the significant activity of secondary amines, it is expected that their incorporations by genetic code expansion will be further applied in the creation of artificial enzymes with designed activity.^61-64

Acknowledgements

We would like to thank EPSRC (EP/L027240/1) for the MS resources and Cardiff School of Chemistry for a PhD studentship to Thomas L. Williams. We would like to thank for the financial support provided by BBSRC (BB/T015799/1), Leverhulme Trust (RPG-2017-195) and Royal Society (RG170187) for Louis Y. P. Luk. We would like to thank Professor Dr Jason Chin at the MRC Laboratory of Molecular Biology for MbPylRS plasmid and Professor Dr Rudolf K Allemann at Cardiff University for the DHFR and TbADH plasmids. We also thank Professor Dr Anthony Green at the University of Manchester for the valuable discussion during manuscript preparation.

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Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

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