Artificial Metalloenzymes for Enantioselective Catalysis: The Phenomenon of Protein Accelerated Catalysis
J. Organomet. Chem. 2004, 689, 4868-4871, 10.1016/j.jorganchem.2004.09.032
We report on the phenomenon of protein-accelerated catalysis in the field of artificial metalloenzymes based on the non-covalent incorporation of biotinylated rhodium–diphosphine complexes in (strept)avidin as host proteins. By incrementally varying the [Rh(COD)(Biot-1)]+ vs. (strept)avidin ratio, we show that the enantiomeric excess of the produced acetamidoalanine decreases slowly. This suggests that the catalyst inside (strept)avidin is more active than the catalyst outside the host protein. Both avidin and streptavidin display protein-accelerated catalysis as the protein embedded catalyst display 12.0- and 3.0-fold acceleration over the background reaction with a catalyst devoid of protein. Thus, these artificial metalloenzymes display an increase both in activity and in selectivity for the reduction of acetamidoacrylic acid.
Ligand type: Cyclooctadiene; DiphenylphosphineHost protein: Streptavidin (Sav)Optimization: ChemicalMax TON: ---ee: 94PDB: ---Notes: Reduction of acetamidoacrylic acid. 3.0-fold protein acceleration.
Ligand type: Cyclooctadiene; DiphenylphosphineHost protein: Avidin (Av)Optimization: ChemicalMax TON: ---ee: 39PDB: ---Notes: Reduction of acetamidoacrylic acid. 12.0-fold protein acceleration.
Artificial Metalloenzymes: (Strept)avidin as Host for Enantioselective Hydrogenation by Achiral Biotinylated Rhodium-Diphosphine Complexes
J. Am. Chem. Soc. 2004, 126, 14411-14418, 10.1021/ja0476718
We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium−diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium−diphosphine catalyst precursor and the host proteins was determined at neutral pH: log Ka = 7.7 for avidin (pI = 10.4) and log Ka = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 °C with a 1% catalyst loading.
Ligand type: PhosphineHost protein: Streptavidin (Sav)Optimization: Chemical & geneticMax TON: ---ee: 94PDB: ---Notes: ---
A Site-Selective Dual Anchoring Strategy for Artificial Metalloprotein Design
J. Am. Chem. Soc. 2004, 126, 10812-10813, 10.1021/ja046908x
Introducing nonnative metal ions or metal-containing prosthetic groups into a protein can dramatically expand the repertoire of its functionalities and thus its range of applications. Particularly challenging is the control of substrate-binding and thus reaction selectivity such as enantioselectivity. To meet this challenge, both non-covalent and single-point attachments of metal complexes have been demonstrated previously. Since the protein template did not evolve to bind artificial metal complexes tightly in a single conformation, efforts to restrict conformational freedom by modifying the metal complexes and/or the protein are required to achieve high enantioselectivity using the above two strategies. Here we report a novel site-selective dual anchoring (two-point covalent attachment) strategy to introduce an achiral manganese salen complex (Mn(salen)), into apo sperm whale myoglobin (Mb) with bioconjugation yield close to 100%. The enantioselective excess increases from 0.3% for non-covalent, to 12.3% for single point, and to 51.3% for dual anchoring attachments. The dual anchoring method has the advantage of restricting the conformational freedom of the metal complex in the protein and can be generally applied to protein incorporation of other metal complexes with minimal structural modification to either the metal complex or the protein.
Metal: MnLigand type: SalenHost protein: Myoglobin (Mb)Anchoring strategy: CovalentReaction: SulfoxidationMax TON: 3.9ee: 51PDB: 1MBONotes: Sperm whale myoglobin
De Novo Design of Catalytic Proteins
Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11566-11570, 10.1073/pnas.0404387101
The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O2-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k cat/K M = 1,500 M·1·min·1) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.
Ligand type: Amino acidHost protein: Due FerriAnchoring strategy: DativeReaction: Alcohol oxidationMax TON: >100ee: ---PDB: ---Notes: kcat/KM ≈ 1540 M-1*min-1
Hybridization of Modified-Heme Reconstitution and Distal Histidine Mutation to Functionalize Sperm Whale Myoglobin
J. Am. Chem. Soc. 2004, 126, 436-437, 10.1021/ja038798k
To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D·2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D·2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.
Ligand type: Double winged protoporphyrin IXHost protein: Myoglobin (Mb)Anchoring strategy: ReconstitutionReaction: Peroxidase activityMax TON: ---ee: ---PDB: ---Notes: ---
Metal-Assembled Modular Proteins: Toward Functional Protein DesignReview
Acc. Chem. Res. 2004, 10.1021/ar960245+
Metal-assembled parallel helix-bundle proteins have been used to investigate electron transfer through α-helical structures. Fermi Golden Rule distance dependence of electron transfer rates was established in a family of designed metalloproteins, and the contribution of intrahelical hydrogen bonding to the matrix tunneling element was explored. The first steps toward the design of functional proteins using dynamic combinatorial assembly of α-helical structural elements are described.
New Activities of a Catalytic Antibody with a Peroxidase Activity: Formation of Fe(II)–RNO Complexes and Stereoselective Oxidation of Sulfides
Eur. J. Biochem. 2004, 271, 1277-1283, 10.1111/j.1432-1033.2004.04032.x
In order to estimate the size of the cavity remaining around the heme of the 3A3–microperoxidase 8 (MP8) hemoabzyme, the formation of 3A3–MP8–Fe(II)‐nitrosoalkane complexes upon oxidation of N‐monosubstituted hydroxylamines was examined. This constituted a new reaction for hemoabzymes and is the first example of fully characterized Fe(II)–metabolite complexes of antibody–porphyrin. Also, via a comparison of the reactions with N‐substituted hydroxylamines of various size and hydrophobicity, antibody 3A3 was confirmed to bring about a partial steric hindrance on the distal face of MP8. Subsequently, the influence of the antibody on the stereoselectivity of the S‐oxidation of sulfides was examined. Our results showed that MP8 alone and the antibody–MP8 complex catalyze the oxidation of thioanisole by H2O2 and tert‐butyl hydroperoxide, following a peroxidase‐like two‐step oxygen‐transfer mechanism involving a radical–cation intermediate. The best system, associating H2O2 as oxidant and 3A3–MP8 as a catalyst, in the presence of 5% tert‐butyl alcohol, led to the stereoselective S‐oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer. This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes.
Ligand type: PorphyrinHost protein: Antibody 3A3Optimization: ---Reaction: SulfoxidationMax TON: 82ee: 45PDB: ---Notes: ---
Peroxidase Activity of Cationic Metalloporphyrin-Antibody Complexes
Chem. - Eur. J. 2004, 10, 6179-6186, 10.1002/chem.200305692
Peroxidase activity of a complex of water‐soluble cationic metalloporphyrin with anti‐cationic porphyrin antibody is reported. Antibody 12E11G, which was prepared by immunization with a conjugate of 5‐(4‐carboxyphenyl)‐10,15,20‐tris(4‐methylpyridyl)porphine iodide (3MPy1C), bound to tetramethylpyridylporphyrin iron complex (FeIII–TMPyP) with the dissociation constant of 2.6×10−7 M. The complex of antibody 12E11G with FeIII–TMPyP catalyzed oxidation of pyrogallol, catechol, and guaiacol. A Lineweaver–Burk plot for the oxidation of pyrogallol catalyzed by the FeIII–TMPyP–antibody complex showed Km=8.6 mM and kcat=680 min−1. Under the same conditions, Km and kcat for horseradish peroxidase (HRP) were 0.8 mM and 1750 min−1, respectively. Although the binding interaction of the antibody to the substrates was one order lower than that of native HRP, the peroxidase activity of this system was in the same order of magnitude as that of HRP.
Ligand type: PorphyrinHost protein: Antibody 12E11GAnchoring strategy: AntibodyOptimization: ---Reaction: Peroxidase activityMax TON: ---ee: ---PDB: ---Notes: ---