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Cavity size engineering of a #-barrel protein generates efficient biohybrid catalysts for olefin metathesis Alexander Richard Grimm, Daniel F. Sauer, Mehdi D. Davari, Leilei Zhu, Marco Bocola, Shunsuke Kato, Akira Onoda, Takashi Hayashi, Jun Okuda, and Ulrich Schwaneberg ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03652 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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ACS Catalysis

Cavity size engineering of a β-barrel protein generates efficient biohybrid catalysts for olefin metathesis Alexander R. Grimm,†,║ Daniel F. Sauer,‡,║ Mehdi D. Davari,† Leilei Zhu,†,┴ Marco Bocola,† Shunsuke Kato,§ Akira Onoda,§ Takashi Hayashi,§ Jun Okuda,*,‡ and Ulrich Schwaneberg*,†,# †

Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen,

Germany ‡

Institute of Inorganic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen,

Germany §

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1

Yamadaoka, Suita 565-0871, Japan #

DWI - Leibniz-Institute for Interactive Materials, Forckenbeckstrasse 50, D-52074 Aachen,

Germany

Incorporation of a synthetic metal catalyst into a protein scaffold yields a biohybrid catalyst, with a remarkable performance in aqueous media and the broad reaction scope of organometallic

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catalysts. A major challenge for efficient catalysis is the design of the interface between the protein scaffold and the metal catalyst. Until now, protein scaffolds have primarily been engineered by exchanging individual amino acids to anchor metal catalysts and alter their immediate environment. Here, cavity size engineering of the β-barrel protein nitrobindin was performed by duplicating multiple β-strands to generate an expanded variant. The approach of cavity size engineering enabled covalent incorporation of bulky catalysts at excellent coupling efficiencies and yielded excellent conversions in olefin metathesis including ring-closing metathesis, ring-opening metathesis polymerization and cross metathesis (conversions up to 99% and TONs up to 10,000).

Keywords: biohybrid catalysis, protein engineering, β-barrel proteins, Grubbs-Hoveyda type catalysts, olefin metathesis

1.

Introduction

By incorporating synthetic metal catalysts into protein scaffolds, so-called biohybrid catalysts have been generated1-3. The value of the respective protein scaffold lies in the defined arrangement of amino acids in the protein scaffold, providing a chiral environment and a robust second coordination sphere around the synthetic catalyst. Importantly, it is possible to enhance or alter specificity and selectivity via protein engineering4-6. Standard protein engineering methods enable the exchange of amino acids at each position within the protein with the nineteen other proteinogenic amino acids. Biohybrid catalysts capable of catalyzing various reaction types have been reported and are summarized in excellent reviews1, 4-12.


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β-Barrel-based scaffolds have recently proved to be robust and desirable scaffolds for biohybrid catalysts. For instance, through site directed mutagenesis the β-barrel protein nitrobindin (NB) was functionalized as a scaffold for a Rh(cp)(cod) catalyst13. One variant (NB4) inverted the stereoselectivity of the poly(phenylacetylene) product from 91% cis to 82% trans14. More recently, we reported a series of highly active biohybrid catalysts for ring-opening metathesis polymerization (ROMP) in water based on Grubbs-Hoveyda (GH) type catalysts coupled to another nitrobindin variant (NB11)15. One main challenge in the generation of such biohybrid catalysts is the productive incorporation of the bulky metal catalyst. Until now, the strategy to overcome this challenge was to extend the spacer length15-17. The latter ensures high coupling efficiencies, but reduces interactions between the metal catalyst and the second coordination sphere provided by the protein scaffold. Here we introduce a new biohybrid catalyst protein scaffold design strategy, which we applied to generate an expanded nitrobindin variant (NB4exp) by introducing two additional β-strands and empowering NB4exp to incorporate bulky GH-type catalysts within its enlarged cavity. Introducing additional β-strands has to the best of our knowledge so far only been reported to improve compound fluxes through membrane proteins in polymer vesicles for the β-barrel protein FhuA (an iron transporter)18. 2.

Results

Coupling efficiencies were determined for three GH-type catalysts (C1, C2, C3) with carbon spacers of varying length. The structural integrity of the resulting biohybrid catalysts (NB4expC1, NB4expC2, NB4expC3) was confirmed by CD spectroscopy. Catalytic performance was determined for N,N-diallyltosylamide and 2,2-diallylpropane-1,3-diol in RCM,


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for two norbornene derivatives in ROMP, and for allyl alcohol, styrene and p-methoxystyrene in CM. Structural Modeling of Protein Scaffold NB4exp. In a previous study, we reported on how enlarging the NB cavity by site-directed mutagenesis had improved the coupling efficiency of the resulting variant with GH-type catalysts15. Encouraged by this result, we set out to design an expanded NB4 variant (NB4exp) to efficiently incorporate bulky GH-type catalysts via shorter spacers. To expand the NB4 cavity, we aimed to add two additional β-strands to the originally 10 stranded β-barrel via duplication of existing β-strands18. The YASARA Structure software suite was used to model the duplication of two β-strands15, 19-21 of NB4 (PDB: 3WJB)14 (see Figure 1 and experimental section for a detailed description; duplicated β-strands 6 to 8; 29 amino acids: Q104 to S132). The N- and C-terminus of the duplicated segment were intentionally positioned within β-stands rather than in loops (Figure 1) to ensure strong interactions between the β-strands (Figures S1-S4, Tables S1-S8 in the Supporting Information; Figure S5: NB4exp sequence in the Supporting Information)22.


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Figure 1. Comparison of the NB4exp model (b) with the crystal structure of NB4 (a). a) NB4 crystal structure14 (PDB: 3WJB) with a reported cavity volume of 855 Å3; b) NB4exp with a computationally determined cavity volume of 1389 Å3 (performed using HotSpot Wizard server v2.0). Duplicated β-strands are shown in orange. Using the HotSpot Wizard server v2.023-24, the cavity volume of NB4exp was determined to be 1389 Å3. The cavity volume of NB414 had previously been reported to be 855 Å3. Since the GHtype catalysts were reported to be up to 795 Å3 in size15, their incorporation into NB4exp seemed promising in contrast to NB4. Coupling Efficiency of NB4exp with GH-Type Catalysts. In order to embed the GH-type catalysts within the β-barrel and not outside or at the surface15 the spacer length was shortened stepwise (C3, C2, C1). The spacer length strongly influences the efficiency of coupling GH-type catalysts in β-barrel proteins and is usually chosen to minimize interactions of the metal catalysts with the protein environment22. The calculated cavity volume


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was probed for three GH-type catalysts (C1: 752 Å3, C2: 768 Å3, C3: 795 Å3) to explore the experimentally available cavity size in NB4 (855 Å3) and NB4exp (1389 Å3). All three GH-type catalysts C1-C3 were covalently anchored to the cysteine C96 of NB4exp via thiol-maleimide “click” reaction (Scheme 1). Sufficient coupling efficiencies up to 90% were observed (coupling efficiencies in Table S9 with ICP-AES results in Table S10 in the Supporting Information; Position of C96 highlighted in Figure S6 in the Supporting Information; Models of NB4exp coupled to GH-type catalysts C1-C3 shown in Figures S7-S9 respectively and selected distances between C96 and Ru atom shown in Table S11 in the Supporting Information). Even with the C1 variant, a high coupling of 90% was achieved (Scheme 1). ESI-TOF MS analysis indicated the attachment of the catalyst to the protein scaffold by covalent anchoring (Figures S10-S13, Table S12 in the Supporting Information). The UV/vis spectra of the biohybrid catalysts showed a new absorption band around 380 nm assigned to the MLCT of the metal catalyst (Figure S14 in the Supporting Information)25. NB4 was unable to serve as a protein scaffold for C1 and C2 spacers15.

Scheme 1. Coupling of GH-type catalysts with varied spacer length (C1-C3) to C96 of NB4exp.


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Structural Characterization of NB4exp. The structural integrity of the NB4exp protein scaffold was confirmed by CD spectroscopy at ambient temperature in buffer (10 mM Tris, pH 7.5; Figure S15 in the Supporting Information). Additionally, structural comparison and alignment to 23 representative 12 β-stranded barrels was performed and supports structural integrity of NB4exp (Figure S16 in the Supporting Information). After proving that NB4exp has a β-barrel fold, CD spectroscopy measurements were performed to analyze thermal (Figure 2) and pH resistance (Figure S17 in the Supporting Information). The thermal resistance measurements in the range between 4 °C and 96 °C showed that NB4exp has a Tm value of 45.3 °C, which is 6.2 °C lower than that of NB4 (51.5 °C). The temperatures used for the various olefin metathesis reactions performed (up to 40 °C) were chosen to be below the Tm value of NB4exp. Heating NB4 and NB4exp above their Tm values led to nearly quantitative and instant precipitation of the proteins.


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Figure 2. Temperature-dependent CD spectroscopy. a) Minimum of CD curves plotted against temperature; b) 3D graph of temperature-dependent CD spectra. NB4 and NB4exp have a similar pH resistance in the pH range of 6-10 in which the olefin metathesis reactions were performed (Figure S17 in the Supporting Information). Additionally, the structural integrity of the biohybrid catalysts NB4expC1-C3 under reaction conditions (5 mM MES, 200 mM NaCl, pH 6) was confirmed through CD spectroscopy (Figures S18-S20 in the Supporting Information). The CD spectra of all NB4exp-based biohybrid catalysts showed no change up to 40 °C. SEC was used to investigate whether NB4exp forms dimers in solution as previously determined for NB4 by SEC15 and crystallography14. SEC elution profiles indicated that in contrast to NB4, NB4exp is monomeric under reaction conditions (Figure S21 in the Supporting Information). Catalytic Performance of NB4exp GH-Type Biohybrid Catalysts. The catalytic performance of the NB4exp-based biohybrid catalysts was determined with olefins for all three basic metathesis types – ring-closing metathesis (RCM), ring-opening metathesis polymerization (ROMP) and cross metathesis (CM). In order to investigate the influence of cavity size on catalytic performance, the corresponding NB4-based biohybrid catalysts and a commercially available, water-soluble GH-type catalyst, AquaMet (no protein scaffold), were employed (see Table 1 for molecular structure). The catalytic activity of the protein-free C1-C3 catalysts was < 10% conversion due to the poor solubility in the aqueous media. Table 1. Selected RCM results of diallyltosylamide 1 and diol 3.


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Entrya

Catalyst

Protein

Substrate

Conv. [%]b

TONb

1

AquaMet

NB4/NB4exp

1

0

-

2

AquaMet

-

1

41

41

3

-

NB4/NB4exp

1

0

-

4

C3

NB4

1

0

-

5

C3

NB4exp

1

35

35

6

C2

NB4exp

1

35

35

7

C1

NB4exp

1

16

16

8

AquaMet

NB4/NB4exp

3

0

-

9

AquaMet

-

3

> 99

100

10c

AquaMet

-

3

70

210

11

-

NB4/NB4exp

3

0

-

12

C3

NB4

3

69

69

13

C3

NB4exp

3

> 99

100

14

C2

NB4exp

3

> 99

100

15c

C2

NB4exp

3

98

296

16

C1

NB4exp

3

45

45

a

Catalyst loading is related to the metal content determined by ICP-AES. b Determined by 1H NMR spectroscopy. c Catalyst loading: 0.33 mol %. All reactions were performed in duplicate. ∆conv. = ±3%


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RCM reaction was performed with the commonly used and water-insoluble substrate N,Ndiallyltosylamide 1 to yield the 2,5-dihydropyrrole derivative 2. Surprisingly NB4 precipitated upon addition of the substrate 1. As a result, product formation could not be detected. The metatheases based on NB4exp did not precipitate upon addition of amide 1, in contrast to the NB4 variant (Table 1, entry 4). In addition to the amide 1, the water-soluble diol substrate 3 was converted to cyclopentene 4 via RCM. Unexpectedly, the metathease based on NB4 did not precipitate upon addition of substrate 3 and converted it to 69% (Table 1, entry 12). Full conversion was achieved with NB4expC3 with a TON of 100 (Table 1, entry 13). The apo-protein did not show any conversion for substrates 1 and 3 (Table 1, entries 3 and 11). Ring-opening metathesis polymerization (ROMP) of the water-soluble 7-oxanorbornene derivative 5 with the NB4exp-based biohybrid catalysts yielded polymers with high molecular weights (Mn up to 750,000 g/mol) and narrow molecular weight distributions (PDI down to 1.21) (Table 2). We had previously reported the incorporation of GH-type catalysts into the smaller cavity of NB4 and the larger cavity of NB11 and how the larger cavity of NB11 was too small to efficiently incorporate the catalysts with short spacers (C1 and C2)15. Notably, in general the obtained catalytic activities with C1 and C2 were poor when compared to C3, which can very likely be attributed to increased interactions between the metathesis catalyst and amino acid side chains in the β-barrel protein (NB4exp) (Table 2, entries 1-3)15. In general, NB4expC2 and NB4expC1 outperformed the corresponding NB11-based biohybrid catalysts (cf. Table 2 entries 6-7 with entries 2-3). NB4expC3 showed an increased catalytic activity in ROMP by approximately one order of magnitude compared to NB4C3 (cf.


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Table 2, entry 5 with entry 4). Compared to NB11C3, the catalytic activity was within the same range, but the molecular weight of the product was approximately four-fold increased for NB4expC2. Furthermore, the catalytic activity of NB4expC1 was three-fold higher when compared to that of NB4C3. Interestingly, with NB4expC1 GPC analysis revealed a polymer with slightly decreased molecular weight compared to NB4expC2/3, but also an oligomeric fraction (dimer, trimer and tetramer). NB4expC1 showed a lower conversion compared to the biohybrid catalysts with longer spacers. All biohybrid catalysts based on NB4exp showed an increased TON in ROMP of norbornene 5 compared to the protein-free water soluble metathesis catalyst AquaMet (Table 2, entry 8), which is structurally closely related. Interestingly, a more than 10-fold increase for the initial rate was observed for the catalyst C2 in the NB4exp protein scaffold (NB4expC2) when compared to the AquaMet catalyst without protein scaffold (kobs(NB4expC2) = 0.39 h-1; kobs(AquaMet) = 0.03 h-1; Figures S22-S23 in the Supporting Information). AquaMet did not show any conversion at all when it was supplemented to NB4/NB4exp and not anchored within the cavity of NB4/NB4exp (Table 2, entry 9). The improvement in comparison to NB4 was also observed with a second, water-soluble and charged norbornene derivative (Table S13 in the Supporting Information). Cross metathesis (CM) was performed with terminal olefins. Table 2. ROMP of 7-oxanorbornene derivative 5.

Entrya

Catalyst

Protein

Conversionb [%]

TON

Mnc x 103

PDIc


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115

C3

NB11

78

9700

180

1.05

215

C2

NB11

19

2100

n.d.

n.d.

315

C1

NB11

99c

100

5

C2

NB4exp

7

> 99c

100

6

C1

NB4exp

7

45c

45

7

AquaMet

-

9

98d

98

8

C3

NB4

9

50d

50

9

C3

NB4exp

9

> 99d

100

10

C2

NB4exp

9

> 99d

100

11

C1

NB4exp

9

45d

45

12

AquaMet

-

11

94d

94

13

C3

NB4

11

45d

45

14

C3

NB4exp

11

> 99d

100

15

C2

NB4exp

11

> 99d

100

16

C1

NB4exp

11

40d

40

Substrate

a

[substrate] = 0.05 M; catalyst loading is related to the metal content determined by ICP-AES. Determined by 1H NMR spectroscopy. All reactions were performed in duplicate. ∆conv. = ±3%. c E/Z = 20/1. d E/Z = 99/1. ∆E/Z = ±1%.

b

3.

Discussion


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The outlined protein scaffold design strategy based on cavity size engineering proved in combination with spacer length variation to be an excellent strategy to tune the synthetic performance of biohybrid catalysts. By duplicating two β-strands of the originally 10 β-stranded NB4 scaffold, the NB4exp variant was generated with an approximately 1.6 times larger cavity volume. Direct comparison of the CD spectra of NB4exp and NB4 for both thermal and pH resistance revealed no significant differences. MD simulations revealed low RMSD (root-mean-square-deviation of the common core) of less than 2 Å between 23 existing protein structures (PDBs: 1FW3, 1ILD, 1ILZ, 1IMO, 2QOM, 3KVN, 3AEH, 2GR8, 3FID, 3ML3, 1UYN, 2WJQ , 1QD5 , 1WP1, 1EK9, 1TLW, 1YC9, 1UYO, 2WJR, 1QD6, 1TLY, 1FW2, 1TLZ; see Figure S16) and NB4exp reveals that structures are very similar to predicted structure of NB4exp, indicating the stability of predicted structure. Visual inspection of aligned structures shows the complete alignment of β-strands, suggesting similar overall barrel shape and β-barrel diameter for 12-stranded β-barrels. The larger cavity of NB4exp improved the coupling efficiency with bulky GH-type catalysts using the maleimide anchoring strategy and enabled the incorporation of the C1 GH-type catalyst to the NB4exp scaffold. In essence, cavity size is fundamental for efficient coupling and high catalytic activity of biohybrid catalysts. To the best of our knowledge, the presented artificial metathease is the first shown to be capable of catalyzing all three basic types of metathesis RCM, ROMP, and CM. The broad reaction scope highlights the system’s attractiveness for transformations in aqueous media. Furthermore, the NB4exp scaffold enabled RCM reactions that could not be performed with the smaller NB4. In the RCM of the benchmark substrate N,N-diallyltosylamide 1, NB4expC2/3 compared favorably to other artificial metatheases, exhibiting increased activity (this work: TON


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(NB4expC3) = 35; Ward et al.: TON (hCAII) = 2826, TON (Avi) = 2016; Hilvert et al.: TON (MjHSP) = 2527; Klein Gebbink et al.: TON (lipase) = 2017; Matsuo et al.: TON (αchymotrypsin) = 425). In the RCM of the diol 3 with the longer C3 spacer, NB4exp performed better compared to NB4. Furthermore, NB4exp bearing shorter spacers showed excellent (C2) to moderate (C1) activity. The decreased activity using NB4expC1 was explained by a loss in flexibility of the catalyst within the cavity. The NB4expC2 outperformed the water soluble AquaMet catalyst with a catalyst loading of 0.33 mol % (Table 1, entries 10 and 15). Compared to NB1115 or to the metathease based on streptavidin28, the activity of NB4expC2 was comparable. In the ROMP of 3,4-bis(methoxymethyl)-7-oxanorbornene 5, the NB4expC1-C3 biohybrid catalysts showed improved activity (TON up to 10,000) compared to NB4C3. The increased cavity size of NB4exp is regarded as the reason for the observed increase in activity. NB4expC3 also showed a 20-fold increased activity compared to the metathease based on FhuA (this work: TON (NB4expC3) = 10,000; TON (FhuA) = 555). Compared to the nitrobindin mutant NB11 coupled to the C3 catalyst, the activity of NB4expC3 was in the same range, but the molecular weight of the polymer product was four times higher (TON (NB11) = 9,700). Regarding the comparison of the performance of the different biohybrid catalysts, it should be noted that the optimized reaction conditions for the metatheases differed in the reports mentioned above. A possible explanation for the lower conversion and molecular weight of product 6 observed with NB4expC1 is that the GH-type catalyst is situated deeper within the protein scaffold with the C1 spacer (Table S11, compare entries 6 with 7-9). This could lead to a blockage of the cavity by the growing polymer, and thereby decrease the polymerization rate. Alternatively, backbiting of the polymer chain could take place, with the hydrophobic polymer forming a coil within the cavity.


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In comparison to water-soluble GH-type catalysts with a PEGylated backbone29, the structure of the NB4exp-based biohybrid catalyst in water is well-defined due to the protein scaffold. This also contributes to the increased activity and broader substrate scope mentioned above. Compared to small molecular catalysts for metathesis in water30-31, such as the AquaMet catalyst employed in this report32, the activity of the NB4exp-based biohybrid catalysts was equivalent or increased. The protein scaffold keeps the active site highly water-soluble. Furthermore, the sensitive ruthenium methylidene intermediate seems to be stabilized by the hydrophobic protein scaffold, leading to increased TON and high conversions. 4.

Conclusion

In summary, the here reported β-barrel expanded biohybrid catalysts are the first biohybrid catalysts shown to be active in all three basic metathesis reaction types – ring-closing metathesis (RCM), ring-opening metathesis polymerization (ROMP), and cross metathesis (CM). The design strategy can likely be transferred to other β-barrel scaffolds (FhuA, streptavidin) to generate cavity sizes that match the sterical demands of synthetic catalysts. The latter will catalyze the convergence of chemical and biological catalysts to fully explore the catalytic potential of biohybrid catalysts, which are especially attractive for synthetic reactions like olefin metathesis that are not naturally catalyzed by enzymes. Furthermore, thanks to the emergence of fluorescence-based high-throughput screening systems, the exploration of directed biohybrid catalyst evolution will likely further increase the performance of biohybrid catalysts (e.g. RCM to produce the fluorescent umbelliferone, a standard screening compound in directed enzyme evolution). AUTHOR INFORMATION


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Corresponding Author *U.S.: email, [email protected]. *J.O.: email, [email protected]. Present Addresses ┴

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th

Avenue, Tianjin 300308, China. Author Contributions All authors have given approval to the final version of the manuscript. ║These authors contributed equally. Funding Sources This work was partially supported by JSPS KAKENHI Grant Number JP17H05370 to A.O and JSPS KAKENHI Grant Number JP15H05804 to T.H. Notes The authors declare no competing financial interest. ASSOCIATED CONTENT Supporting Information. General comments, experimental section, genetic construction of NB4exp, NB4exp amino acid sequence, characterization of coupling and coupled biohybrid catalysts, characterization of catalyzed reactions. The Supporting Information is available free of charge on the ACS Publications website. ACKNOWLEDGMENTS


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We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft (DFG) through the International Research Training Group “Selectivity in Chemo- and Biocatalysis” (SeleCa), the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297), JSPS KAKENHI Grant Number JP15KT0144 to A.O., JSPS KAKENHI Grant Number JP 17H05370 in Innovative Areas “Coordination Asymmetry” to A.O., JSPS KAKENHI Grant Number JP15H05804 in Innovative Areas “Precisely Designed Catalysts with Customized Scaffolding” to T.H., and Umicore, Frankfurt (Dr. A. Doppiu), for a generous gift of ruthenium precursor. The “His-eGFP-Helix-TEV” 29 kDa eGFP variant used for SEC was kindly provided by Lina Weber. Marcus Arlt is acknowledged for valuable discussions. Dr. T. Tsujimoto and Prof. H. Uyama are acknowledged for their assistance with GPC measurements at Osaka University. S.K. acknowledges support from the Interactive Material Science Cadet program (IMSC). ABBREVIATIONS Avi, avidin; C1, Grubbs-Hoveyda type catalyst 1; C2, Grubbs-Hoveyda type catalyst 2; C3, Grubbs-Hoveyda type catalyst 3; CD, circular dichroism; CM, cross metathesis; eGFP, enhanced green fluorescent protein; ESI-TOF MS, electrospray ionization time-of-flight mass spectrometry; FhuA, ferric hydroxamate uptake protein component A; GH, Grubbs-Hoveyda; GPC, gel permeation chromatography; hCAII, human carbonic anhydrase II; ICP-AES, inductively coupled plasma atomic emission spectroscopy; MjHSP, M. jannashii small heat shock protein; MLCT, metal-to-ligand charge-transfer; Mn, number average molar mass; NB, nitrobindin; NB11, nitrobindin variant 11; NB4, nitrobindin variant 4; NB4exp, nitrobindin variant 4 expanded; PDI, polydispersity index; RCM, ring-closing metathesis; Rh(cp)(cod), rhodium complex with cyclopentadiene and cyclooctadiene ligands; ROMP, ring-opening


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