26 publications
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Abiological Catalysis by Artificial Haem Proteins Containing Noble Metals in Place of Iron
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Nature 2016, 534, 534-537, 10.1038/nature17968
Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites1,4,5. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein6,7,8. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes9,10. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.
Metal: IrHost protein: Myoglobin (Mb)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Myoglobin (Mb)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
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An Artificial Metalloenzyme with the Kinetics of Native Enzymes
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Science 2016, 354, 102-106, 10.1126/science.aah4427
Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene insertions into C–H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.
Metal: IrHost protein: Cytochrome P450 (CYP119)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Cytochrome P450 (CYP119)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
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A Well-Defined Osmium–Cupin Complex: Hyperstable Artificial Osmium Peroxygenase
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J. Am. Chem. Soc. 2017, 139, 5149-5155, 10.1021/jacs.7b00675
Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.
Metal: OsLigand type: Amino acidHost protein: TM1459 cupinAnchoring strategy: Metal substitutionOptimization: GeneticNotes: Exclusively cis dihydroxylation product obtained
Metal: OsLigand type: Amino acidHost protein: TM1459 cupinAnchoring strategy: Metal substitutionOptimization: GeneticNotes: Exclusively cis dihydroxylation product obtained
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Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes
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ACS Cent. Sci. 2017, 3, 302-308, 10.1021/acscentsci.6b00391
Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C–H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.
Metal: IrHost protein: Cytochrome P450 (CYP119)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: Selectivity for cis product (cis/trans = 90:1)
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Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus Aureus
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J. Biol. Chem. 1967, n/a
A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mp which accompanies deoxyribonucleic acid and RNA hy- drolysis. Initial velocities are proportional to enzyme con- centration over a 70-fold range. The enzyme has greater aflinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is be- tween 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca+f is required. The inhibitory effect of high Ca+f concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr+f, while Fe++ and C&f cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca+f or Sr+f suggest that these substrates are hy- drolyzed by the same or closely related regions on the en- zyme. Enzyme activity toward DNA and RNA is strongly in- hibited by 5’-phosphoryl (not by 2’- or 3’-phosphoryl) deriva- tives of deoxyadenylic, adenylic, and deozythymidylic acids, and deozythymidine 3’,5’-diphosphate is the most po- tent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These tidings are consistent with the known action of the enzyme (cleavage of the 5’-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the afhnity toward the constituent bases of these nucleic acids.
Metal: SrLigand type: Amino acidHost protein: Nuclease from S. aureusAnchoring strategy: Metal substitutionOptimization: ---Notes: PMID 4290246; DNA cleavage
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Catalytic Water Oxidation by Iridium-Modified Carbonic Anhydrase
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Chem. - Asian J. 2018, 13, 334-341, 10.1002/asia.201701543
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme with a Zn cofactor coordinated to trigonal histidine imidazole moieties in a tetrahedral geometry. Removal of the Zn cofactor in CA and subsequent binding of Ir afforded CA[Ir]. Under mild and neutral conditions (30 °C, pH 7), CA[Ir] exhibited water‐oxidizing activity with a turnover frequency (TOF) of 39.8 min−1, which is comparable to those of other Ir‐based molecular catalysts. Coordination of Ir to the apoprotein of CA is thermodynamically preferred and is associated with an exothermic energy change (ΔH) of −10.8 kcal mol−1, which implies that the CA apoprotein is stabilized by Ir binding. The catalytic oxygen‐evolving activity of CA[Ir] is displayed only if Ir is bound to CA, which functions as an effective biological scaffold that activates the Ir center for catalysis. The results of this study indicate that the histidine imidazoles at the CA active site could be exploited as beneficial biological ligands to provide unforeseen biochemical activity by coordination to a variety of transition‐metal ions.
Metal: IrLigand type: Amino acidHost protein: Bovine carbonic anhydrase (CA)Anchoring strategy: Metal substitutionOptimization: ChemicalNotes: Sodium periodate as sacrificial oxidant. TOF at pH 7 and 30°C is 39.8 min-1.
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Chemoselective, Enzymatic C−H Bond Amination Catalyzed by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor
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J. Am. Chem. Soc. 2017, 139, 1750-1753, 10.1021/jacs.6b11410
Cytochrome P450 enzymes have been engineered to catalyze abiological C–H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C–H bonds over competing reduction of the azide substrate to a sulfonamide. Here we report that P450s derived from a thermophilic organism and containing an iridium porphyrin cofactor (Ir(Me)-PIX) in place of the heme catalyze enantioselective intramolecular C−H bond amination reactions of sulfonyl azides. These reactions occur with chemoselectivity for insertion of the nitrene units into C−H bonds over reduction of the azides to the sulfonamides that is higher and with substrate scope that is broader than those of enzymes containing iron porphyrins. The products from C−H amination are formed in up to 98% yield and ∼300 TON. In one case, the enantiomeric excess reaches 95:5 er, and the reactions can occur with divergent site selectivity. The chemoselectivity for C–H bond amination is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX CYP119 displaying these properties were identified rapidly by evaluating CYP119 mutants containing Ir(Me)-PIX in cell lysates, rather than as purified enzymes. This study sets the stage to discover suitable enzymes to catalyze challenging C–H amination reactions.
Metal: IrHost protein: Cytochrome P450 (CYP119)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Cytochrome P450 (CYP119)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
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Direct Hydrogenation of Carbon Dioxide by an Artificial Reductase Obtained by Substituting Rhodium for Zinc in the Carbonic Anhydrase Catalytic Center. A Mechanistic Study
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ACS Catal. 2015, 5, 5397-5409, 10.1021/acscatal.5b00185
Recently, a new artificial carbonic anhydrase enzyme in which the native zinc cation has been replaced with a Rh(I) has been proposed as a new reductase that is able to efficiently catalyze the hydrogenation of olefins. In this paper, we propose the possible use of this modified enzyme in the direct hydrogenation of carbon dioxide. In our theoretical investigation, we have considered different reaction mechanisms such as reductive elimination and σ-bond metathesis. In addition, the release of the formic acid and the restoring of the catalytic cycle have also been studied. Results show that the σ-bond metathesis potential energy surface lies below the reactant species. The rate-determining step is the release of the product with an energy barrier of 12.8 kcal mol–1. On the basis of our results, we conclude that this artificial enzyme can efficiently catalyze the conversion of CO2 to HCOOH by a direct hydrogenation reaction.
Metal: RhLigand type: Amino acidHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: ---Notes: Computational study of the reaction mechanism of the formation of HCOOH from CO2
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Going Beyond Structure: Nickel-Substituted Rubredoxin as a Mechanistic Model for the [NiFe] Hydrogenases
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J. Am. Chem. Soc. 2018, 140, 10250-10262, 10.1021/jacs.8b05194
Well-defined molecular systems for catalytic hydrogen production that are robust, easily generated, and active under mild aqueous conditions remain underdeveloped. Nickel-substituted rubredoxin (NiRd) is one such system, featuring a tetrathiolate coordination environment around the nickel center that is identical to the native [NiFe] hydrogenases and demonstrating hydrogenase-like proton reduction activity. However, until now, the catalytic mechanism has remained elusive. In this work, we have combined quantitative protein film electrochemistry with optical and vibrational spectroscopy, density functional theory calculations, and molecular dynamics simulations to interrogate the mechanism of H2 evolution by NiRd. Proton-coupled electron transfer is found to be essential for catalysis. The coordinating thiolate ligands serve as the sites of protonation, a role that remains debated in the native [NiFe] hydrogenases, with reduction occurring at the nickel center following protonation. The rate-determining step is suggested to be intramolecular proton transfer via thiol inversion to generate a NiIII–hydride species. NiRd catalysis is found to be completely insensitive to the presence of oxygen, another advantage over the native [NiFe] hydrogenase enzymes, with potential implications for membrane-less fuel cells and aerobic hydrogen evolution. Targeted mutations around the metal center are seen to increase the activity and perturb the rate-determining process, highlighting the importance of the outer coordination sphere. Collectively, these results indicate that NiRd evolves H2 through a mechanism similar to that of the [NiFe] hydrogenases, suggesting a role for thiolate protonation in the native enzyme and guiding rational optimization of the NiRd system.
Metal: NiLigand type: Amino acidHost protein: Rubredoxin (Rd)Anchoring strategy: Metal substitutionOptimization: GeneticNotes: TOF = 149 s-1
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Intramolecular C(sp3)-H Amination of Arylsulfonyl Azides with Engineered and Artificial Myoglobin-Based Catalysts
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Bioorg. Med. Chem. 2014, 22, 5697-5704, 10.1016/j.bmc.2014.05.015
The direct conversion of aliphatic CH bonds into CN bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following the recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp3)H amination reaction, we have explored here the CH amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were prepared to investigate the effect of these structural changes on the catalytic activity and selectivity of these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable CH amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that CH bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of CH amination catalysts.
Metal: MnHost protein: Myoglobin (Mb)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
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Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein
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J. Am. Chem. Soc. 2020, 142, 5338-5354, 10.1021/jacs.0c00333
Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving–Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C–H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C–H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.
Ligand type: Amino acidHost protein: R2-like ligand-binding oxidase (R2lox)Anchoring strategy: Metal substitutionOptimization: ---Notes: PDB: 6QK0, 6QJV, 6QK2, 6QK1
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Manganese-Substituted Carbonic Anhydrase as a New Peroxidase
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Chem. - Eur. J. 2006, 12, 1587-1596, 10.1002/chem.200501413
Carbonic anhydrase is a zinc metalloenzyme that catalyzes the hydration of carbon dioxide to bicarbonate. Replacing the active‐site zinc with manganese yielded manganese‐substituted carbonic anhydrase (CA[Mn]), which shows peroxidase activity with a bicarbonate‐dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, (CA[Mn]) catalyzed the efficient oxidation of o‐dianisidine with kcat/KM=1.4×106 m−1 s−1, which is comparable to that for horseradish peroxidase, kcat/KM=57×106 m−1 s−1. CA[Mn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E=5 for p‐chlorostyrene) in the presence of an amino‐alcohol buffer, such as N,N‐bis(2‐hydroxyethyl)‐2‐aminoethanesulfonic acid (BES). This enantioselectivity is similar to that for natural heme‐based peroxidases, but has the advantage that CA[Mn] avoids the formation of aldehyde side products. CA[Mn] degrades during the epoxidation limiting the yield of the epoxidations to <12 %. Replacement of active‐site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site‐directed mutagenesis decreased the enantioselectivity demonstrating that the active site controls the enantioselectivity of the epoxidation.
Metal: MnLigand type: Amino acidHost protein: Bovine carbonic anhydrase II (CA)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: MnLigand type: Amino acidHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: PDB ID 4CAC = Structure of Zn containing hCAII
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Metal Ion Dependent Binding of Sulphonamide to Carbonic Anhydrase
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Nature 1967, 214, 193-194, 10.1038/214193a0
ACETAZOLAMIDE (2-acetylamino-1,3,4-thiadiazole-5-sulphonamide, ‘Diamox’) is the most potent known inhibitor of the zinc enzyme carbonic anhydrase. This communication reports the direct demonstration that binding of acetazolamide to human carbonic anhydrase requires the presence of a metal ion at the active site and that binding depends on the species of divalent metal ion present. Zinc (II) and cobalt (II) ions are the only ions which induce the formation of very stable acetazolamide carbonic anhydrase complexes and are also the ions which most effectively catalyse the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate. Metal-binding monodentate ions, CN−, HS−, OCN−, and N3−, known as effective carbonic anhydrase inhibitors, compete for the acetazolamide binding site of the zinc enzyme.
Metal: CoLigand type: Amino acidHost protein: Human carbonic anhydraseAnchoring strategy: Metal substitutionOptimization: ---Notes: CO2 hydration
Metal: CoLigand type: Amino acidHost protein: Human carbonic anhydraseAnchoring strategy: Metal substitutionOptimization: ---Notes: Ester cleavage
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Metal Substitution in Thermolysin: Catalytic Properties of Tungstate Thermolysin in Sulfoxidation with H2O2
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Can. J. Chem. 2002, 80, 622-625, 10.1139/v02-082
The catalytic Zn2+ ion was extracted from thermolysin, which had been covalently bound to Eupergit C. The apo-enzyme incorporated the oxometallate anions MoO42, SeO42, and WO42 with partial restoration of the proteolytic activity. Tungstate thermolysin was moderately active in the sulfoxidation of thioanisole by hydrogen peroxide, whereas its activity towards phenylmercaptoacetophenone, which was designed to bind well in the active site of thermolysin, was much higher.
Metal: WLigand type: Amino acidHost protein: ThermolysinAnchoring strategy: Metal substitutionOptimization: ChemicalNotes: ---
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Multifunctional Nanoenzymes from Carbonic Anhydrase Skeleton
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Process Biochem. 2018, 72, 71-78, 10.1016/j.procbio.2018.06.005
Carbonic anhydrase (carbonic dehydratase) (CA) is a metalloenzyme that contains zinc (Zn2+) ion in its active site. CA catalyzes the reversible conversion of carbon dioxide and water to bicarbonate and protons. Zn2+ ions, which are present in the active site of the enzyme, interact with the substrate molecules directly and cause catalytic effect. In this study, a nano-enzyme system was designed in aqueous solutions at room temperature and under nitrogen atmosphere to use the CA enzyme without any pre-treatment and deformation in its structure. The novel concept ANADOLUCA (AmiNo Acid (monomer) Decorated and Light Underpinning Conjugation Approach) was used for this process, nano CA enzyme of size 93 nm was synthesized. The activity of the synthesized nano CA was measured following the change in absorbance during the conversion of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm for a period of 10 min at 25 °C compared with free CA enzyme. Km and Vmax values for nano CA enzyme were found to be 0.442 mM and 1.6 × 10−3 mM min-1, respectively, whereas Km and Vmax values for free CA were found to be 0.471 mM and 1.5 × 10−3 mM min-1, respectively. In addition to these, the Zn2+ ion present in the active site of the nano CA enzyme was replaced by rodium metal. This nanorodium-substituted CA has been investigated as a new reductase enzyme for the stereoselective hydrogenation of olefins. Then, the Zn2+ ion in the active site of the nano CA enzyme was replaced with manganese metal to enhance the enzyme structure, thereby gaining characteristics of peroxidase. This newly synthesized nano manganese-substituted CA enzyme was investigated for its role as a peroxidase, which could be an alternative for hydrogen peroxidases.
Metal: ZnLigand type: Amino acidHost protein: Carbonic anhydrase (CA)Anchoring strategy: Metal substitutionOptimization: ChemicalNotes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydrolysis of 4-nitrophenyl acetate.
Metal: RhLigand type: Amino acidHost protein: Carbonic anhydrase (CA)Anchoring strategy: Metal substitutionOptimization: ChemicalNotes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydration of styrene.
Metal: MnLigand type: Amino acidHost protein: Carbonic anhydrase (CA)Anchoring strategy: Metal substitutionOptimization: ChemicalNotes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Oxidation of styrene.
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Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase
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J. Phys. Chem. Lett. 2015, 6, 3731-3736, 10.1021/acs.jpclett.5b01750
A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20–100 s–1 at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.
Metal: NiLigand type: TetrathiolateHost protein: Rubredoxin (Rd)Anchoring strategy: Metal substitutionOptimization: ---Notes: ---
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Polymer Enzyme Conjugates as Chiral Ligands for Sharpless Dihydroxylation of Alkenes in Organic Solvents
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ChemBioChem 2015, 16, 83-90, 10.1002/cbic.201402339
Count Os in: We report organosoluble artificial metalloenzymes, generated from poly(2‐methyl‐oxazoline) enzyme conjugates and osmate as a promising new catalytic system for the dihydroxylation of alkenes in organic media.
Metal: OsLigand type: Amino acidHost protein: LaccaseAnchoring strategy: Metal substitutionOptimization: ChemicalNotes: ---
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Protein Secondary-Shell Interactions Enhance the Photoinduced Hydrogen Production of Cobalt Protoporphyrin IX
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Chem. Commun. 2014, 50, 15852-15855, 10.1039/c4cc06700b
Hydrogen is an attractive fuel with potential for production scalability, provided that inexpensive, efficient molecular catalysts utilizing base metals can be developed for hydrogen production. Here we show for the first time that cobalt myoglobin (CoMyo) catalyzes hydrogen production in mild aerobic conditions with turnover number of 520 over 8 hours. Compared to free Co-protoporphyrin IX, incorporation into the myoglobin scaffold results in a 4-fold increase in photoinduced hydrogen production activity. Engineered variants in which specific histidine resides in proximity of the active site were mutated to alanine result in modulation of the catalytic activity, with the H64A/H97A mutant displaying activity 2.5-fold higher than wild type. Our results demonstrate that protein scaffolds can augment and modulate the intrinsic catalytic activity of molecular hydrogen production catalysts.
Metal: CoLigand type: PorphyrinHost protein: Myoglobin (Mb)Anchoring strategy: Metal substitutionOptimization: GeneticNotes: ---
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Rare Earth Metal Ions as Probes of Calcium Binding Sites in Proteins: Neodynium Acceleration of the Activation of Trypsinogen
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J. Biol. Chem. 1970, n/a
The rate of activation of the conversion of trypsinogen to trypsin has been found to be greatly accelerated by the neodymium(III) ion. The similarity of this process to the calcium(II) ion activation suggests that both metal ions bind at identical sites in trypsinogen. The rate of activation in the presence of the neodymium ion is much greater than that of the calcium ion, probably reflecting the increased stability constant of the neodymium-protein complex. In contrast to the calcium ion, however, neodymium(III) can be scrutinized by a variety of spectral and magnetic techniques which should reveal information concerning the calcium ion binding sites in proteins. Since the chemistry and the range of sires of the rare earth metal ions are so similar to that of the calcium ion, it is suggested that generally these ions should make good replacement ions for probing the calcium ion binding sites of proteins and enzymes.
Metal: NdLigand type: Amino acidHost protein: TrypsinAnchoring strategy: Metal substitutionOptimization: ---Notes: PMID 5484822
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Reengineering Cyt b562 for Hydrogen Production: A Facile Route to Artificial Hydrogenases
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Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 598-603, 10.1016/j.bbabio.2015.09.001
Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.
Metal: CoLigand type: PorphyrinHost protein: Cytochrome b562Anchoring strategy: Metal substitutionOptimization: GeneticNotes: ---
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Regioselective Hydroformylation of Styrene Using Rhodium-Substituted Carbonic Anhydrase
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ChemCatChem 2010, 2, 953-957, 10.1002/cctc.201000159
CA confidential: Replacing the active‐site zinc in carbonic anhydrase (CA) by rhodium forms a new enzymatic catalyst for cofactor‐free hydroformylation of styrene with syn gas. Unlike free rhodium, this rhodium–protein hybrid, [Rh]–CA, is regioselective (8.4:1) for linear over branched aldehyde product, which is a 40‐fold change in regioselectivity compared to free rhodium.
Metal: RhHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: GeneticNotes: PDB ID 4CAC = Structure of Zn containing hCAII
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Semisynthetic and Biomolecular Hydrogen Evolution Catalysts
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Inorg. Chem. 2016, 55, 467-477, 10.1021/acs.inorgchem.5b02054
There has been great interest in the development of stable, inexpensive, efficient catalysts capable of reducing aqueous protons to hydrogen (H2), an alternative to fossil fuels. While synthetic H2 evolution catalysts have been in development for decades, recently there has been great progress in engineering biomolecular catalysts and assemblies of synthetic catalysts and biomolecules. In this Forum Article, progress in engineering proteins to catalyze H2 evolution from water is discussed. The artificial enzymes described include assemblies of synthetic catalysts and photosynthetic proteins, proteins with cofactors replaced with synthetic catalysts, and derivatives of electron-transfer proteins. In addition, a new catalyst consisting of a thermophilic cobalt-substituted cytochrome c is reported. As an electrocatalyst, the cobalt cytochrome shows nearly quantitative Faradaic efficiency and excellent longevity with a turnover number of >270000.
Metal: CoLigand type: PorphyrinHost protein: Cytochrome c552Anchoring strategy: Metal substitutionOptimization: GeneticNotes: Electrocatalysis
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Stereoselective Hydrogenation of Olefins Using Rhodium-Substituted Carbonic Anhydrase—A New Reductase
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Chem. - Eur. J. 2009, 15, 1370-1376, 10.1002/chem.200801673
One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA‐[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site‐directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA‐[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis‐ over trans‐stilbene by about 20:1. This enzyme is the first cofactor‐independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme‐catalyzed reactions.
Metal: RhLigand type: CODHost protein: Bovine carbonic anhydrase II (CA)Anchoring strategy: Metal substitutionOptimization: GeneticNotes: ---
Metal: RhLigand type: CODHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: GeneticNotes: PDB ID 4CAC = Structure of Zn containing hCAII
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Studies on the Oxidase Activity of Copper (II) Carboxypeptidase A
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J. Chem. Soc., Chem. Commun. 1976, 830, 10.1039/C39760000830
Copper(II) carboxypeptidase A catalyses the oxidation of ascorbic acid and this reaction is inhibited by α-benzylsuccinate, a known inhibitor of the thiolesterase action of the copper enzyme; the pH dependencies of kcat and kcat/Km are similar near pH 7 to those seen for the peptidase and esterase activities of native carboxypeptidase A.
Metal: CuLigand type: Amino acidHost protein: Carboxypeptidase AAnchoring strategy: Metal substitutionOptimization: ---Notes: Oxidation of vitamin C
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Transforming Carbonic Anhydrase into Epoxide Synthase by Metal Exchange
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ChemBioChem 2006, 7, 1013-1016, 10.1002/cbic.200600127
Enantioselective epoxidation of styrene was observed in the presence of manganese‐containing carbonic anhydrase as catalyst. The probable oxygen‐transfer reagent is peroxymonocarbonate, which has a structural similarity with the hydrogenocarbonate substrate of the natural reaction. Styrene was chosen as the enzyme possesses a small hydrophobic cavity close to the active site.
Metal: MnLigand type: Amino acidHost protein: Bovine carbonic anhydrase II (CA)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: MnLigand type: Amino acidHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
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Unnatural Biosynthesis by an Engineered Microorganism with Heterologously Expressed Natural Enzymes and an Artificial Metalloenzyme
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Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3
Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.
Notes: TON in vivo of (-)-carvone, WITHOUT limonene biosynthetic genes