A Highly Active Biohybrid Catalyst for Olefin Metathesis in Water: Impact of a Hydrophobic Cavity in a β-Barrel Protein
ACS Catal. 2015, 5, 7519-7522, 10.1021/acscatal.5b01792
A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.
Metal: RuLigand type: CarbeneReaction: Olefin metathesisMax TON: 9900ee: ---PDB: ---Notes: ROMP (cis/trans: 48/52)
Metal: RuLigand type: CarbeneReaction: Olefin metathesisMax TON: 100ee: ---PDB: ---Notes: RCM
A Rhodium Complex-Linked β-Barrel Protein as a Hybrid Biocatalyst for Phenylacetylene Polymerization
Chem. Commun. 2012, 48, 9756, 10.1039/C2CC35165J
Our group recently prepared a hybrid catalyst containing a rhodium complex, Rh(Cp)(cod), with a maleimide moiety at the peripheral position of the Cp ligand. This compound was then inserted into a β-barrel protein scaffold of a mutant of aponitrobindin (Q96C) via a covalent linkage. The hybrid protein is found to act as a polymerization catalyst and preferentially yields trans-poly(phenylacetylene) (PPA), although the rhodium complex without the protein scaffold normally produces cis PPA.
Optimization: ---Reaction: Phenylacetylene polymerizationMax TON: ---ee: ---PDB: ---Notes: ---
Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme
ACS Catal. 2021, 11, 5079-5087, 10.1021/acscatal.1c00134
Directed evolution has helped enzyme engineering to remarkable successes in the past. A main challenge in directed evolution is to find the most suitable starting point, that is, an enzyme that allows maximum “evolvability”. Consisting of a synthetic cofactor embedded in a protein scaffold, artificial metalloenzymes (ArMs) are reminiscent of rough-hewn ancestral metalloproteins and thus could provide an evolutionarily clean slate. Here, we report the design and directed evolution of an ArM with peroxidase-like properties based on the nitrobindin variant, NB4. After identifying a suitable artificial metal cofactor, two rounds of directed evolution were sufficient to elevate the ArM’s activity to levels akin to those of some natural peroxidases (up to kcat = 14.1 s–1 and kcat/Km = 52,800 M–1 s–1). A substitution to arginine in the distal cofactor environment (position 76) was the key to boost the peroxidase activity. Molecular dynamics simulations reveal a remarkable flexibility in the distal site of the NB4 scaffold that is absent in the nitrobindin wildtype and which allows the unrestricted movement of the catalytically important Arg76. In addition to the oxidation of the common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol), the ArM proved efficient in the decolorization of three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5) and was amenable to several rounds of ArM recycling.
Ligand type: PorphyrinAnchoring strategy: SupramolecularOptimization: Chemical & geneticReaction: OxidationMax TON: ---ee: ---PDB: ---Notes: kcat = 14.1 s−1 and kcat/Km = 52,800 M−1 s −1
Construction of a Hybrid Biocatalyst Containing a Covalently-Linked Terpyridine Metal Complex within a Cavity of Aponitrobindin
J. Inorg. Biochem. 2016, 158, 55-61, 10.1016/j.jinorgbio.2015.12.026
A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB–1. Binding of Cu2 +, Zn2 +, or Co2 + ion to the tpy ligand in NB–1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu2 +-bound NB–1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu2 +–tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.
Metal: CuLigand type: TerpyridineOptimization: ---Reaction: Diels-Alder reactionMax TON: ---ee: ---PDB: ---Notes: ---
Directed Evolution of a Cp*RhIII‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification
ChemBioChem 2021, 22, 679-685, 10.1002/cbic.202000681
Directed evolution of Cp*RhIII-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.
Ligand type: CpOptimization: GeneticReaction: CycloadditionMax TON: ---ee: ---PDB: ---Notes: ---
Manganese Terpyridine Artificial Metalloenzymes for Benzylic Oxygenation and Olefin Epoxidation
Tetrahedron 2014, 70, 4245-4249, 10.1016/j.tet.2014.03.008
New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.
Ligand type: Poly-pyridineReaction: Peroxidation or oxygenationMax TON: 19.2ee: ---PDB: 3EMMNotes: ---
Ligand type: Poly-pyridineReaction: EpoxidationMax TON: 19.8ee: ---PDB: 3EMMNotes: ---
Photoinduced Hydrogen Evolution Catalyzed by a Synthetic Diiron Dithiolate Complex Embedded within a Protein Matrix
ACS Catal. 2014, 4, 2645-2648, 10.1021/cs500392e
The hydrogen-evolving diiron complex, (μ-S)2Fe2(CO)6 with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)2Fe2(CO)6 core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H2 via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C.
Metal: FeLigand type: Carbonyl; DithiolateOptimization: ---Reaction: H2 evolutionMax TON: 130ee: ---PDB: ---Notes: ---
Rhodium-Complex-Linked Hybrid Biocatalyst: Stereo-Controlled Phenylacetylene Polymerization within an Engineered Protein Cavity
ChemCatChem 2014, n/a-n/a, 10.1002/cctc.201301055
The incorporation of a Rh complex with a maleimide moiety into the cavity of the nitrobindin β‐barrel scaffold by a covalent linkage at the 96‐position (Cys) provides a hybrid biocatalyst that promotes the polymerization of phenylacetylene. The appropriate structural optimization of the cavity by mutagenesis enhances the stereoselectivity of the polymer with a trans content of 82 % at 25 °C and pH 8.0. The X‐ray crystal structure of one of the hybrid biocatalysts at a resolution of 2.0 Å reveals that the Rh complex is located in the β‐barrel cavity without any perturbation to the total protein structure. Crystal structure analysis and molecular modeling support the fact that the stereoselectivity is enhanced by the effective control of monomer access to the Rh complex within the limited space of the protein cavity.
Optimization: GeneticReaction: Phenylacetylene polymerizationMax TON: ---ee: ---PDB: 3WJCNotes: ---