The Experts below are selected from a list of 73104 Experts worldwide ranked by ideXlab platform
Brian Kuhlman - One of the best experts on this subject based on the ideXlab platform.
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catalysis by a de novo zinc mediated protein interface implications for Natural Enzyme evolution and rational Enzyme engineering
Biochemistry, 2012Co-Authors: David R Edwards, Brian KuhlmanAbstract:Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein–protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 105 and a kcat/KM of 630 M–1 s–1 and 4-nitrophenyl phosphate with a rate acceleration of 104 and a kcat/KM of 14 M–1 s–1. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein–protein interactions are well-suited for creating Enzyme active sites. This discovery has implications for protein evolution and engineering: from an evo...
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catalysis by a de novo zinc mediated protein interface implications for Natural Enzyme evolution and rational Enzyme engineering
Biochemistry, 2012Co-Authors: Bryan S Der, David R Edwards, Brian KuhlmanAbstract:Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating Enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of Enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.
David R Edwards - One of the best experts on this subject based on the ideXlab platform.
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catalysis by a de novo zinc mediated protein interface implications for Natural Enzyme evolution and rational Enzyme engineering
Biochemistry, 2012Co-Authors: David R Edwards, Brian KuhlmanAbstract:Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein–protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 105 and a kcat/KM of 630 M–1 s–1 and 4-nitrophenyl phosphate with a rate acceleration of 104 and a kcat/KM of 14 M–1 s–1. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein–protein interactions are well-suited for creating Enzyme active sites. This discovery has implications for protein evolution and engineering: from an evo...
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catalysis by a de novo zinc mediated protein interface implications for Natural Enzyme evolution and rational Enzyme engineering
Biochemistry, 2012Co-Authors: Bryan S Der, David R Edwards, Brian KuhlmanAbstract:Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating Enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of Enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.
Bryan S Der - One of the best experts on this subject based on the ideXlab platform.
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catalysis by a de novo zinc mediated protein interface implications for Natural Enzyme evolution and rational Enzyme engineering
Biochemistry, 2012Co-Authors: Bryan S Der, David R Edwards, Brian KuhlmanAbstract:Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating Enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of Enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.
Frances H Arnold - One of the best experts on this subject based on the ideXlab platform.
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design and evolution of Enzymes for non Natural chemistry
Green and Sustainable Chemistry, 2017Co-Authors: Stephan C Hammer, Anders M Knight, Frances H ArnoldAbstract:Enzymes are used in biocatalytic processes for the efficient and sustainable production of pharmaceuticals, fragrances, fine chemicals, and other products. Most bioprocesses exploit chemistry found in nature, but we are now entering a realm of biocatalysis that goes well beyond. Enzymes have been engineered to catalyze reactions previously only accessible with synthetic catalysts. Because they can be tuned by directed evolution, many of these new biocatalysts have been shown to perform abiological reactions with high activity and selectivity. We discuss recent examples, showcase catalyst improvements achieved using directed evolution, and comment on some current and future implications of non-Natural Enzyme evolution for sustainable chemical synthesis.
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the nature of chemical innovation new Enzymes by evolution
Quarterly Reviews of Biophysics, 2015Co-Authors: Frances H ArnoldAbstract:I describe how we direct the evolution of non-Natural Enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/Enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N–H insertion, C–H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new Enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists’ favorite transformations with the exquisite selectivity and tunability of Enzymes.
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enantioselective Enzyme catalyzed aziridination enabled by active site evolution of a cytochrome p450
ACS central science, 2015Co-Authors: Christopher C Farwell, Ruijie K Zhang, John A Mcintosh, Todd K Hyster, Frances H ArnoldAbstract:One of the greatest challenges in protein design is creating new Enzymes, something evolution does all the time, starting from existing ones. Borrowing from nature’s evolutionary strategy, we have engineered a bacterial cytochrome P450 to catalyze highly enantioselective intermolecular aziridination, a synthetically useful reaction that has no Natural biological counterpart. The new Enzyme is fully genetically encoded, functions in vitro or in whole cells, and can be optimized rapidly to exhibit high enantioselectivity (up to 99% ee) and productivity (up to 1,000 catalytic turnovers) for intermolecular aziridination, demonstrated here with tosyl azide and substituted styrenes. This new aziridination activity highlights the remarkable ability of a Natural Enzyme to adapt and take on new functions. Once discovered in an evolvable Enzyme, this non-Natural activity was improved and its selectivity tuned through an evolutionary process of accumulating beneficial mutations.
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improved cyclopropanation activity of histidine ligated cytochrome p450 enables the enantioselective formal synthesis of levomilnacipran
Angewandte Chemie, 2014Co-Authors: Jane Z Wang, Christopher C Farwell, Hans Renata, Nicole E Peck, Pedro S Coelho, Frances H ArnoldAbstract:Engineering Enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting Enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a Natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 Enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This Enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-Natural Enzyme activity.
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efficient screening of fungal cellobiohydrolase class i Enzymes for thermostabilizing sequence blocks by schema structure guided recombination
Protein Engineering Design & Selection, 2010Co-Authors: Pete Heinzelman, Russell S Komor, Arvind Kanaan, Philip A Romero, Shannon Mohler, Christopher D Snow, Frances H ArnoldAbstract:We describe an efficient SCHEMA recombination-based approach for screening homologous Enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) Enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent Enzymes and just 32 ‘monomeras’ containing a single block from a homologous Enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610–5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of Natural Enzyme sequence space for desirable amino acid blocks.
Jian Chen - One of the best experts on this subject based on the ideXlab platform.
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ratiometric surface enhanced raman scattering immunosorbent assay of allergenic proteins via covalent organic framework composite material based nanozyme tag triggered raman signal turn on and amplification
Analytical Chemistry, 2019Co-Authors: Yiyun Su, Jian Chen, Guang Chen, Honglun Wang, Panxue Wang, Di Wu, Na Hu, Guoliang Li, Yongning WuAbstract:The exploration of nanomaterials with mimic Enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional Natural Enzyme tag in an Enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au–S bond and electrostatic force to further produce a Raman “hot spot”. Meanwhile, the Raman signal of 4-nitrothiophenol ...
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ratiometric surface enhanced raman scattering immunosorbent assay of allergenic proteins via covalent organic framework composite material based nanozyme tag triggered raman signal turn on and amplification
Analytical Chemistry, 2019Co-Authors: Jian Chen, Guang Chen, Honglun Wang, Panxue Wang, Haoyu HanAbstract:The exploration of nanomaterials with mimic Enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional Natural Enzyme tag in an Enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au–S bond and electrostatic force to further produce a Raman “hot spot”. Meanwhile, the Raman signal of 4-nitrothiophenol ...
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Enzyme Engineering and Industrial Bioprocess
Current Developments in Biotechnology and Bioengineering, 2019Co-Authors: Chen Deng, Xueqin Lv, Zhu Jiang, Jian Chen, Tingting Huang, Guocheng DuAbstract:Abstract Enzymes play an important role in life activities due to their specific and efficient catalytic action. Nowadays, Enzymes derived from microorganisms have been extensively developed to serve various human needs, such as brewing, enzymatic conversion, disease diagnosis and treatment, drug production, environmental pollutant removal, etc. However, Natural Enzymes are often very expensive, and most of them are difficult to be effectively utilized because they are very “delicious.” In recent years, the development of structural biology and genetic manipulation technology has enabled scientists to effectively reform and design Enzyme molecules with “purposes,” which promotes the development of molecular Enzyme engineering. In summary, molecular Enzyme engineering is a discipline that uses the genetic engineering and protein engineering methods and techniques to study the cloning and expression of Enzyme genes, the relationship between the structure and function of Enzyme proteins, and the redesign and orientation of Enzymes to further improve excellent Enzyme or new functional Enzyme. This chapter mainly introduces Enzyme engineering research from the following three aspects: firstly, the use of genetic engineering technology to produce large amounts of Enzyme preparations; secondly, through molecular site-directed mutagenesis and in vitro molecular-directed evolution of Natural Enzyme protein molecular modification; finally, the industrial application of Enzymes examples.