Recent advances in the design and optimization of artificial metalloenzymes.

Embedding a catalytically competent transition metal into a protein scaffold affords an artificial metalloenzyme (ArM). Such hybrid catalysts display features that are reminiscent of both homogeneous and enzymatic catalysts. Pioneered by Whitesides and Kaiser in the late 1970s, this field of ArMs has expanded over the past two decades, marked by ever-increasing diversity in reaction types, cofactors, and protein scaffolds. Recent noteworthy developments include i) the use of earth-abundant metal cofactors, ii) concurrent cascade reactions, iii) synergistic catalysis, and iv) in vivo catalysis. Thanks to significant progress in computational protein design, ArMs based on de novo-designed proteins and tailored chimeric proteins promise a bright future for this exciting field.

Enhanced Sequence-Activity Mapping and Evolution of Artificial Metalloenzymes by Active Learning.

Tailored enzymes are crucial for the transition to a sustainable bioeconomy. However, enzyme engineering is laborious and failure-prone due to its reliance on serendipity. The efficiency and success rates of engineering campaigns may be improved by applying machine learning to map the sequence-activity landscape based on small experimental data sets. Yet, it often proves challenging to reliably model large sequence spaces while keeping the experimental effort tractable. To address this challenge, we present an integrated pipeline combining large-scale screening with active machine learning, which we applied to engineer an artificial metalloenzyme (ArM) catalyzing a new-to-nature hydroamination reaction. Combining lab automation and next-generation sequencing, we acquired sequence-activity data for several thousand ArM variants. We then used Gaussian process regression to model the activity landscape and guide further screening rounds. Critical characteristics of our pipeline include the cost-effective generation of information-rich data sets, the integration of an explorative round to improve the model's performance, and the inclusion of experimental noise. Our approach led to an order-of-magnitude boost in the hit rate while making efficient use of experimental resources. Search strategies like this should find broad utility in enzyme engineering and accelerate the development of novel biocatalysts.

An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway.

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.

Pnictogen-Bonding Enzymes.

The objective of this study was to create artificial enzymes that capitalize on pnictogen bonding, a s-hole interaction that is essentially absent in biocatalysis.  For this purpose, stibine catalysts were equipped with a biotin derivative and combined with streptavidin mutants to identify an efficient transfer hydrogenation catalyst for the reduction of a fluorogenic quinoline substrate.  Increased catalytic activity from wild-type streptavidin to the best mutants coincides with the depth of the s hole on the Sb(V) center, and the emergence of saturation kinetic behavior.  Michaelis-Menten analysis reveals transition-state recognition in the low micromolar range, more than three orders of magnitude stronger than the millimolar substrate recognition.  Carboxylates preferred by the best mutants contribute to transition-state recognition by hydrogen-bonded ion pairing and anion-π interactions with the emerging pyridinium product.  The emergence of challenging stereoselectivity in aqueous systems further emphasizes compatibility of pnictogen bonding with higher order systems catalysis.

NSPs: chromogenic linkers for fast, selective, and irreversible cysteine modification.

The addition of a sulfhydryl group to water-soluble N-alkyl(o-nitrostyryl)pyridinium ions (NSPs) followed by fast and irreversible cyclization and aromatization results in a stable S-C sp2-bond. The reaction sequence, termed Click & Lock, engages accessible cysteine residues under the formation of N-hydroxy indole pyridinium ions. The accompanying red shift of >70 nm to around 385 nm enables convenient monitoring of the labeling yield by UV-vis spectroscopy at extinction coefficients of ≥2 × 104 M-1 cm-1. The versatility of the linker is demonstrated in the stapling of peptides and the derivatization of proteins, including the modification of reduced trastuzumab with Val-Cit-PAB-MMAE. The high stability of the linker in human plasma, fast reaction rates (k app up to 4.4 M-1 s-1 at 20 °C), high selectivity for cysteine, favorable solubility of the electrophilic moiety and the bathochromic properties of the Click & Lock reaction provide an appealing alternative to existing methods for cysteine conjugation.

C-H functionalization reactions catalyzed by artificial metalloenzymes.

CH functionalization, a promising frontier in modern organic chemistry, facilitates the direct conversion of inert CH bonds into many valuable functional groups. Despite its merits, traditional homogeneous catalysis, often faces challenges in efficiency, selectivity, and sustainability towards this transformation. In this context, artificial metalloenzymes (ArMs), resulting from the incorporation of a catalytically-competent metal cofactor within an evolvable protein scaffold, bridges the gap between the efficiency of enzymatic transformations and the versatility of transition metal catalysis. Accordingly, ArMs have emerged as attractive tools for various challenging catalytic transformations. Additionally, the coming of age of directed evolution has unlocked unprecedented avenues for optimizing enzymatic catalysis. Taking advantage of their genetically-encoded protein scaffold, ArMs have been evolved to catalyze various CH functionalization reactions. This review delves into the recent developments of ArM-catalyzed CH functionalization reactions, highlighting the benefits of engineering the second coordination sphere around a metal cofactor within a host protein.

An artificial nickel chlorinase based on the biotin-streptavidin technology.

Herein, we report on an artificial nickel chlorinase (ANCase) resulting from anchoring a biotinylated nickel-based cofactor within streptavidin (Sav). The resulting ANCase was efficient for the chlorination of diverse C(sp3)-H bonds. Guided by the X-ray analysis of the ANCase, the activity of the artificial chlorinase could be significantly improved. This approach opens interesting perspectives for late-stage functionalization of organic intermediates as it complements biocatalytic chlorination strategies.

Binding Interactions and Inhibition Mechanisms of Gold Complexes in Thiamine Diphosphate-Dependent Enzymes.

Thiamine diphosphate (ThDP)-dependent enzymes possess the unique ability to generate a carbene within their active site. In this study, we sought to harness this carbene to produce a Au(I) N-heterocyclic complex directly in the active site of ThDP enzymes, thereby establishing a novel platform for artificial metalloenzymes. Because direct metalation of ThDP proved challenging, we synthesized a ThDP mimic that acts as a competitive inhibitor with a high affinity (Ki = 1.5 µM). Upon metalation with Au(I), we observed that the complex became a more potent inhibitor (Ki = 0.7 µM). However, detailed analysis of the inhibition mode, native mass spectrometry, and size exclusion experiments revealed that the complex does not bind specifically to the active site of ThDP enzymes. Instead, it exhibits unspecific binding and exceeds the 1:1 stoichiometry. Similar binding patterns were observed for other Au(I) species. These findings prompt an important question regarding the inherent propensity of ThDP enzymes to bind strongly to Au. If this phenomenon holds true, it could pave the way for the development of Au-based drugs targeting these enzymes.

A Co(TAML)-based artificial metalloenzyme for asymmetric radical-type oxygen atom transfer catalysis.

We show that the incorporation of a biotinylated Co(TAML) cofactor within streptavidin enables asymmetric radical-type oxygen atom transfer catalysis with improved activity and enantioselectivity.

Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.

A Customized Bayesian Algorithm to Optimize Enzyme-Catalyzed Reactions.

Design of experiments (DoE) plays an important role in optimizing the catalytic performance of chemical reactions. The most commonly used DoE relies on the response surface methodology (RSM) to model the variable space of experimental conditions with the fewest number of experiments. However, the RSM leads to an exponential increase in the number of required experiments as the number of variables increases. Herein we describe a Bayesian optimization algorithm (BOA) to optimize the continuous parameters (e.g., temperature, reaction time, reactant and enzyme concentrations, etc.) of enzyme-catalyzed reactions with the aim of maximizing performance. Compared to existing Bayesian optimization methods, we propose an improved algorithm that leads to better results under limited resources and time for experiments. To validate the versatility of the BOA, we benchmarked its performance with biocatalytic C-C bond formation and amination for the optimization of the turnover number. Gratifyingly, up to 80% improvement compared to RSM and up to 360% improvement vs previous Bayesian optimization algorithms were obtained. Importantly, this strategy enabled simultaneous optimization of both the enzyme's activity and selectivity for cross-benzoin condensation.

Artificial Metalloenzyme-Catalyzed Enantioselective Amidation via Nitrene Insertion in Unactivated C(sp3)-H Bonds.

Enantioselective C-H amidation offers attractive means to assemble C-N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C-H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)-H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)-H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance.

An Artificial [Fe4S4]-Containing Metalloenzyme for the Reduction of CO2 to Hydrocarbons.

Iron-sulfur clusters have been reported to catalyze various redox transformations, including the multielectron reduction of CO2 to hydrocarbons. Herein, we report the design and assembly of an artificial [Fe4S4]-containing Fischer-Tropschase relying on the biotin-streptavidin technology. For this purpose, we synthesized a bis-biotinylated [Fe4S4] cofactor with marked aqueous stability and incorporated it in streptavidin. The effect of the second coordination sphere provided by the protein environment was scrutinized by cyclic voltammetry, highlighting the accessibility of the doubly reduced [Fe4S4] cluster. The Fischer-Tropschase activity was improved by chemo-genetic means for the reduction of CO2 to hydrocarbons with up to 14 turnovers.

Spiers Memorial Lecture: Shielding the active site: a streptavidin superoxide-dismutase chimera as a host protein for asymmetric transfer hydrogenation.

By anchoring a metal cofactor within a host protein, so-called artificial metalloenzymes can be generated. Such hybrid catalysts combine the versatility of transition metals in catalyzing new-to-nature reactions with the power of genetic-engineering to evolve proteins. With the aim of gaining better control over second coordination-sphere interactions between a streptavidin host-protein (Sav) and a biotinylated cofactor, we engineered a hydrophobic dimerization domain, borrowed from superoxide dismutase C (SOD), on Sav's biotin-binding vestibule. The influence of the SOD dimerization domain (DD) on the performance of an asymmetric transfer hydrogenase (ATHase) resulting from anchoring a biotinylated Cp*Ir-cofactor - [Cp*Ir(biot-p-L)Cl] (1-Cl) - within Sav-SOD is reported herein. We show that, depending on the nature of the residue at position Sav S112, the introduction of the SOD DD on the biotin-binding vestibule leads to an inversion of configuration of the reduction product, as well as a fivefold increase in catalytic efficiency. The findings are rationalized by QM/MM calculations, combined with X-ray crystallography.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions.

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.