Directed Evolution of an Artificial Imine Reductase

Abstract

Artificial metalloenzymes,r esulting from incorporation of ametal cofactor within ahost protein, have received increasing attention in the last decade.The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin-streptavidin technology using as traightforward procedure allowing screening in cell-free extracts.T wo streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines.T he evolved ATHases were stable under biphasic catalytic conditions.T he X-ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor,t hus increasing exposure of the metal to the protein surface and leading to ar eversal of enantioselectivity.T his hypothesis was confirmed by am ultiscale approach based mostly on molecular dynamics and protein-ligand dockings.

Artificial imine reductases: developments and future directions

“…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.…”

“…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.…”

Artificial imine reductases: developments and future directions

“…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.…”

“…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.…”

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

“… 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.…”

An Artificial Hemoprotein with Inducible Peroxidase‐ and Monooxygenase‐Like Activities

“…With the aim to shed light on the molecular events leading to the switchable catalysis, am olecular modelling study was performed as it has now become ak ey player in sustaining ArM design with or without X-ray structures available. [47][48][49][50] First, a series of large Molecular Dynamics (MD) was carried on:i )BH-MnTPP;i i) BH-MnTPP in presence of the triggering His 6 -bA3-2 protein,a nd iii)His 6 -bA3-2i ncluded BH-MnTPP with different possible coordination between the Mn III ion and histidine residues from the His 6 -Tag of bA3-2 (His5, His7, His8 of the His 6 -Tag from the N-terminal protein sequence M 1 R 2 G 3 S 4 H 5 H 6 H 7 H 8 H 9 H 10 ). The MDs served to evaluate the flexibility of the bio-hybrid, the viability of substrate binding cavities for the reactiont ot ake place,a nd finally the best possible axial coordination for the metal throughout a N-His donor.…”

Artificial imine reductases: developments and future directions

“…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.…”

“…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.…”

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

“… 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.…”

An Artificial Hemoprotein with Inducible Peroxidase‐ and Monooxygenase‐Like Activities

“…With the aim to shed light on the molecular events leading to the switchable catalysis, am olecular modelling study was performed as it has now become ak ey player in sustaining ArM design with or without X-ray structures available. [47][48][49][50] First, a series of large Molecular Dynamics (MD) was carried on:i )BH-MnTPP;i i) BH-MnTPP in presence of the triggering His 6 -bA3-2 protein,a nd iii)His 6 -bA3-2i ncluded BH-MnTPP with different possible coordination between the Mn III ion and histidine residues from the His 6 -Tag of bA3-2 (His5, His7, His8 of the His 6 -Tag from the N-terminal protein sequence M 1 R 2 G 3 S 4 H 5 H 6 H 7 H 8 H 9 H 10 ). The MDs served to evaluate the flexibility of the bio-hybrid, the viability of substrate binding cavities for the reactiont ot ake place,a nd finally the best possible axial coordination for the metal throughout a N-His donor.…”

Artificial imine reductases: developments and future directions

“…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.…”

“…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.…”

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

“… 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.…”

An Artificial Hemoprotein with Inducible Peroxidase‐ and Monooxygenase‐Like Activities

“…With the aim to shed light on the molecular events leading to the switchable catalysis, am olecular modelling study was performed as it has now become ak ey player in sustaining ArM design with or without X-ray structures available. [47][48][49][50] First, a series of large Molecular Dynamics (MD) was carried on:i )BH-MnTPP;i i) BH-MnTPP in presence of the triggering His 6 -bA3-2 protein,a nd iii)His 6 -bA3-2i ncluded BH-MnTPP with different possible coordination between the Mn III ion and histidine residues from the His 6 -Tag of bA3-2 (His5, His7, His8 of the His 6 -Tag from the N-terminal protein sequence M 1 R 2 G 3 S 4 H 5 H 6 H 7 H 8 H 9 H 10 ). The MDs served to evaluate the flexibility of the bio-hybrid, the viability of substrate binding cavities for the reactiont ot ake place,a nd finally the best possible axial coordination for the metal throughout a N-His donor.…”