Directed Evolution of an Artificial Imine Reductase

Hestericová, Martina; Heinisch, Tillmann; Alonso‐Cotchico, Lur; Maréchal, Jean‐Didier; Vidossich, Pietro; Ward, Thomas R.

doi:10.1002/ange.201711016

Cited by 12 publications

(10 citation statements)

References 81 publications

(37 reference statements)

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“…The main example of this strategy is the exploitation of the supramolecular biotin-(strept)avidin interaction in which a biotinylated diamine ligand in coordination with a transition metal centre was anchored to a streptavidin mutant and employed as an artificial imine reductase in the stereoselective reduction of imines. [2][3][4][5][6][7] Vancomycin (Van) is a glycopeptide antibiotic used as a last resort to effectively treat methicillinresistant Staphylococcus aureus (MRSA) infections. The mode of action of Van involves the selective binding of the antibiotic to the D-Ala-D-Ala (DADA) sequence of the bacterial cell wall peptidoglycan, [8][9][10][11] leading to an inhibition of cell growth and eventual cell death.…”

Section: Introductionmentioning

confidence: 99%

Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex

et al. 2021

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Based on the supramolecular interaction between vancomycin (Van), an antibiotic glycopeptide, and D-Ala-D-Ala (DADA) dipeptides, a novel class of artificial metalloenzymes was synthesized and characterized. The presence of an iridium(III) ligand at the N-terminus of DADA allowed the use of the metalloenzyme as a catalyst in the asymmetric transfer hydrogenation of cyclic imines. In particular, the type of link between DADA and the metal-chelating moiety was found to be fundamental for inducing asymmetry in the reaction outcome, as highlighted by both computational studies and catalytic results. Using the [IrCp*(m-I)Cl]Cl⊂Van complex in 0.1 M CH3COONa buffer at pH 5, a significant 70% (S) e.e. was obtained in the reduction of quinaldine B.

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Section: Introductionmentioning

confidence: 99%

Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex

et al. 2021

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“…The development of new screening techniques has proven important in allowing faster development and use of methods, such as directed evolution. 53,54,56 Progress in the use of computational modelling to better understand the interactions between the synthetic catalyst and the protein looks to have potential to help with the design of better performing ArMs. 49,60 Immobilisation of artIR-EDs has provided additional ways to improve stability and recovery strategies.…”

Section: Discussionmentioning

confidence: 99%

“…In a pioneering study, 54 directed evolution was carried out on a small library of amino acids located around the iridium catalyst. It was concluded that bulky residues can limit the degrees of freedom of the bound cofactor, increase the interactions between the protein and the metal ion and influence the enantioselectivity observed.…”

Section: Rsc Chemical Biology Reviewmentioning

confidence: 99%

Artificial imine reductases: developments and future directions

et al. 2020

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“… 31 Implementation of this diamide strategy allowed for optimization by directed evolution of an artificial imine reductase, using cell free extracts. 32 The scope of this strategy, however, is limited because (i) diamide is incompatible with some catalysts and (ii) the method is significantly more laborious than traditional in vivo evolution methods, limiting the number of variants that can be practically screened. Alternatively, sequestration of ArMs into chemically distinct compartments offers the opportunity to minimize catalyst poisoning.…”

Section: Compartmentalization and Surface Display Of Arms: Versatile mentioning

confidence: 99%

Artificial Metalloenzymes Based on the Biotin–Streptavidin Technology: Enzymatic Cascades and Directed Evolution

et al. 2019

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ConspectusArtificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin–(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L–1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo.With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of “endowing organometallic chemistry with a genetic memory.” With this goal in mind, we have identified E. coli’s periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype–genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C–H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of t...

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Directed Evolution of an Artificial Imine Reductase

Cited by 12 publications

References 81 publications

Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex

Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex

Artificial imine reductases: developments and future directions

Artificial Metalloenzymes Based on the Biotin–Streptavidin Technology: Enzymatic Cascades and Directed Evolution

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