LmrR:A Privileged Scaffold for Artificial Metalloenzymes

The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism

Repurposed and artificial heme enzymes for cyclopropanation reactions

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed

Directed evolution of artificial metalloenzyme – in vivo catalysis

There is a growing interest in implementing organometallic catalysis in the context of synthetic biology for sustainable production of chemicals. Some of the recent achievements in this field include development of bio-compatible cyclopropanation by Arnold and Balskus groups[1,2]. As a first step towards interfacing microbial metabolism we aim to utilize Artificial metalloenzymes (ArMs) to perform catalysis in the cell to augment cellular bio-synthesis. Integrating ArMs catalyzed reactions in cells also provides a springboard to apply Darwinian evolution to improve the performance of these primordial enzymes[3]. Please click Additional Files below to see the full abstract

Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites

Artificial enzymes, which are hybrids of proteins with abiological catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiological catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiological catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalysed Michael addition reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiological catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes

Artificial Metalloenzymes

Artificial metalloenzymes have emerged as a promising approach to merge the attractive properties of homogeneous catalysis and biocatalysis. The activity and selectivity, including enantioselectivity, of natural metalloenzymes are due to the second coordination sphere interactions provided by the protein. Artificial metalloenzymes aim at harnessing second coordination sphere interactions to create transition metal complexes that display enzyme-like activities and selectivities. In this Review, the various approaches that can be followed for the design and optimization of an artificial metalloenzyme are discussed. An overview of the synthetic transformations that have been achieved using artificial metalloenzymes is provided, with a particular focus on recent developments. Finally, the role that the second coordination sphere plays in artificial metalloenzymes and their potential for synthetic applications are evaluated

Expanding the enzyme universe with genetically encoded unnatural amino acids

The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chemical functionalities in enzymes. In this Perspective, we show how this powerful methodology is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes containing only canonical amino acids

Synergistic Catalysis of Tandem Michael Addition/Enantioselective Protonation Reactions by an Artificial Enzyme

[Image: see text] Enantioselective protonation is conceptually one of the most attractive methods to generate an α-chiral center. However, enantioselective protonation presents major challenges, especially in water. Herein, we report a tandem Michael addition/enantioselective protonation reaction catalyzed by an artificial enzyme employing two abiological catalytic sites in a synergistic fashion: a genetically encoded noncanonical p-aminophenylalanine residue and a Lewis acid Cu(II) complex. The exquisite stereocontrol achieved in the protonation of the transient enamine intermediate is illustrated by up to >20:1 dr and >99% ee of the product. These results illustrate the potential of exploiting synergistic catalysis in artificial enzymes for challenging reactions

Double strand DNA cleavage with a binuclear iron complex

Covalently linking two single strand DNA cleaving agents resulted in a new biomimetic binuclear iron complex capable of effecting oxidative double strand DNA cleavage.