Artificial Metalloenzymes Based on the Biotin–Streptavidin Technology: Challenges and Opportunities

Abstract

The biotin-streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin-(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as "molecular velcro", allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C-H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin-streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified protein samples. This seriously limits the throughput of the optimization process. With the ultimate goal of complementing natural enzymes in the context of synthetic and chemical biology, we outline the milestones required to ultimately implement ArMs within a cellular environment. Indeed, we believe that ArMs may allow signficant expansion of the natural enzymes' toolbox to access new-to-nature reactivities in vivo. With this ambitious goal in mind, we report on our efforts to (i) activate the biotinylated catalyst precursor upon incorporation within streptavidin, (ii) minimize the effect of the cellular environment on the ArM's performance, and (iii) demonstrate the compatibility of ArMs with natural enzymes in cascade reactions.

Opportunities and challenges for combining chemo- and biocatalysis

“…In this case, a biotinylated homogeneous metal catalyst was localized within the streptavidin core. This compartmentalization concept led to the development of biocompatible artificial catalysts for hydrogenations, transfer hydrogenations of ketones and enones, Suzuki cross-coupling reactions, C-H activations and C-C ligations via cross-metathesis 82,83 . Recently, Jeschek et al demonstrated the power of the streptavidin concept by developing a Hoveyda-Grubbs second-generation Ru catalyst containing metalloenzyme for in vivo metathesis in the periplasm of E. coli 84 .…”

Peroxide Activation Regulated by Hydrogen Bonds within Artificial Cu Proteins

“…This approach relies on the strong binding of biotinylated metal complexes to the Sav host, leading to the precise localization of the synthetic metal cofactors within a protein environment. 17 Structure-function relationships between the local environment and the stability of the Cu–OOH complex were evaluated using a series of Sav variants in which individual H-bonds were systematically deleted.…”

Tandem Reactions Combining Biocatalysts and Chemical Catalysts for Asymmetric Synthesis

“…An intense area of research has been the generation of artificial metalloenzymes by incorporating metal catalysts into protein scaffolds such as streptavidin [76], bovine serum albumin, and apomyoglobin [77]. With the aid of computational, molecular and structural biology, functional artificial metalloenzymes have been developed by de novo design or protein redesign processes [78].…”

“…This method sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-proteins scaffolds and unnatural metal cofactors for various abiological transformations. techniques and examples of various artificial metalloenzymes [51,76,[78][79][80][81][82][83][84]. To avoid duplication, we will only highlight the major accomplishments since 2015 in terms of tandem catalysis.…”