Evolution of an artificial allylic alkylase based on the biotin-streptavidin technology

The PhD thesis presented here summarizes the work and the scientific effort done in the research group of Prof. Dr. Ward at the University of Basel during the years 2013 – 2017. The Ward group has a long-term knowledge in the design and evolution of artificial metalloenzymes capable of catalyzing reactions including transfer hydrogenation, ring-closing metathesis, C-H activation, Suzuki-coupling and many more. Artificial metalloenzymes are formed by the incorporation of a catalytically active transition-metal complex into a host protein. This allows combining the advantageous features of both homogeneous catalysis and enzyme catalysis. The protein forms a defined reaction environment (i.e. a second coordination sphere) around the metal cofactor. Thus, artificial metalloenzymes can be evolved by chemical modification of the metal cofactor or by genetic engineering of the host protein. In the Ward group often the biotin-streptavidin technology is applied to generate artificial metalloenzymes. This system relies on the ultra-high affinity of the protein streptavidin for the small molecule biotin. Attachment of a biotin-anchor to a transition-metal complex ensures its incorporation into the streptavidin scaffold. In this thesis the design, expression and evolution of an artificial allylic deallocase based on the biotin-streptavidin technology is described. A biotinylated ruthenium complex was synthesized, incorporated into streptavidin and a crystal structure of the resulting artificial metalloenzyme was determined. The activity of the hybrid catalyst in a deallocation reaction was investigated. An O-allyl carbamate caged pro-fluorescent coumarin derivative was deprotected in the presence of the artificial metalloenzyme. The in vitro performance of the artificial allylic deallocase was evolved by genetic modification of the host protein. In a next step, the artificial metalloenzyme was displayed on the surface of E. coli cells. The activity of the hybrid catalyst was further evolved by in vivo screening of several single-site saturation mutagenesis libraries. It was aimed to further increase the throughput of the screening assay by application of a microfluidic system in combination with fluorescence-activated droplet sorting. In a third step, a biogenetic switch based on O-allyl carbamate caged inducer molecules was designed. By the action of the artificial allylic deallocase, the caged inducer was deprotected and subsequently induced the expression of a green fluorescent protein (GFP)-reporter. By substitution of the GFP with another natural protein, a cascade reaction can be envisioned. In parallel, a series of streptavidin mutants with lid-like amino acid structures on top of the biotin-binding vestibule was designed. This approach aimed gaining a better control of the second coordination sphere of the metal cofactor in order to increase the activity and selectivity of the artificial metalloenzyme. In summary, these efforts should allow a straightforward design, expression and evolution of new artificial metalloenzymes for in vivo applications. During the time in the Ward group a deeper knowledge on protein design and expression, molecular biology, synthesis of organometallic cofactors, in vivo catalysis and high-throughput screening based on microfluidics was garnered

Chimeric streptavidins as host proteins for artificial metalloenzymes

The streptavidin scaffold was expanded with well-structured naturally occurring motifs. These chimeric scaffolds were tested as hosts for biotinylated catalysts as artificial metalloenzymes (ArM) for asymmetric transfer hydrogenation, ring-closing metathesis and anion−π catalysis. The additional second coordination sphere elements significantly influence both the activity and the selectivity of the resulting hybrid catalysts. These findings lead to the identification of propitious chimeric streptavidins for future directed evolution efforts of artificial metalloenzymes

Light-driven electron injection from a biotinylated triarylamine donor to [Ru(diimine)3]2+-labeled streptavidin

Electron transfer from a biotinylated electron donor to photochemically generated Ru(III) complexes covalently anchored to streptavidin is demonstrated by means of time-resolved laser spectroscopy. Through site-selective mutagenesis, a single cysteine residue was engineered at four different positions on streptavidin, and a Ru(II) tris-diimine complex was then bioconjugated to the exposed cysteines. A biotinylated triarylamine electron donor was added to the Ru(II)-modified streptavidins to afford dyads localized within a streptavidin host. The resulting systems were subjected to electron transfer studies. In some of the explored mutants, the phototriggered electron transfer between triarylamine and Ru(III) is complete within 10 ns, thus highlighting the potential of such artificial metalloenzymes to perform photoredox catalysis

A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells

E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M-K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970’s. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000’s. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000’s, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors

On-cell catalysis by surface engineering of live cells with an artificial metalloenzyme

Metal-catalyzed chemical transformations performed at the cellular level bear great potential for the manipulation of biological processes. The complexity of the cell renders the use of transition metal chemistry difficult in cellular systems. The delivery of the reactive catalyst and the control of its spatial localization remain challenging. Here we report the surface functionalization of the unicellular eukaryote Chlamydomonas reinhardtii with a tailor-made artificial metalloenzyme for on-cell catalysis. The functionalized cells remain viable and are able to uncage a fluorogenic substrate on their surface. This work leverages cell surface engineering to provide live cells with new-to-nature reactivity. In addition, this operationally simple approach is not genetically encoded and thereby transient, which offers advantages with regard to temporal control, cell viability, and safety. Therefore, and as a feature, the movement of the functionalized cells can be directed by light (via phototaxis), allowing for the three-dimensional localization of catalysts by outside stimuli

Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based on; Escherichia coli; surface-displayed streptavidin (Sav; SD; hereafter). Through the binding of a ruthenium-pianostool cofactor to Sav; SD; , an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase of; in vivo; activity was observed for Sav; SD; S112T-K121G compared to the wild-type Sav; SD; (wt-Sav; SD; ). Finally, the best performing Sav isoforms were purified and tested; in vitro; (Sav; PP; hereafter). For Sav; PP; S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-Sav; PP; . To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of Sav; SD; was determined. For this purpose, crosslinking experiments of; E. coli; cells overexpressing Sav; SD; were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that Sav; SD; is most likely displayed as a monomer on the surface of; E. coli; . We hypothesize that the difference between the; in vivo; and; in vitro; screening results may reflect the difference in the oligomeric state of Sav; SD; vs soluble Sav; PP; (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using; E. coli; surface display

Expanding the Chemical Diversity in Artificial Imine Reductases Based on the Biotin-Streptavidin Technology

We report on the optimization of an artificial imine reductase based on the biotin-streptavidin technology. With the aim of rapidly generating chemical diversity, a novel strategy for the formation and evaluation of biotinylated complexes is disclosed. Tethering the biotin-anchor to the Cp* moiety leaves three free coordination sites on a d6 metal for the introduction of chemical diversity by coordination of a variety of ligands. To test the concept, 34 bidentate ligands were screened and a selection of the 6 best was tested in the presence of 21 streptavidin (Sav) isoforms for the asymmetric imine reduction by the resulting three legged piano stool complexes. Enantiopure α-amino amides were identified as promising bidentate ligands: up to 63 % ee and 190 turnovers were obtained in the formation of 1-phenyl-1,2,3,4-tetrahydroisoquinoline with [IrCp*biotin(L-ThrNH2)Cl]⊂SavWT as a catalyst

Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin–Streptavidin Technology

Artificial metalloenzymes enable the engineering of the reaction microenvironment of the active metal catalyst by modification of the surrounding host protein. We report herein the optimization of an artificial imine reductase (ATHase) based on biotin–streptavidin technology. By introduction of lipophilic amino acid residues around the active site, an 8-fold increase in catalytic efficiency compared with the wild type imine reductase was achieved. Whereas substrate inhibition was encountered for the free cofactor and wild type ATHase, two engineered systems exhibited classical Michaelis–Menten kinetics, even at substrate concentrations of 150 mM with measured rates up to 20 min^–1