Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

Cesaro-Tadic, Sandro; Lagos, Dimitrios; Honegger, Annemarie; Rickard, James H.; Partridge, Lynda J.; Blackburn, G. Michael; Plückthun, Andreas

doi:10.1038/nbt828

Cited by 83 publications

(49 citation statements)

References 44 publications

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“…We anticipate the successful use of the combination of computational design and molecular evolution that we have described here for a wide range of important reactions in the years to come. The challenge of generating new biocatalysts has led to several successful experimental strategies [20][21][22] . In particular, the Kemp elimination comprises a well-defined model for catalysis of proton transfer from carbon-a highly demanding reaction and a rate-determining step in numerous enzymes.…”

Section: Discussionmentioning

confidence: 99%

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Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

1,197

1,237

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The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10 5 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a .200-fold increase in k cat /K m (k cat /K m of 2,600 M 21 s 21 and k cat /k uncat of .10 6 ). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.Naturally occurring enzymes are extraordinarily efficient catalysts 1 . They bind their substrates in a well-defined active site with precisely aligned catalytic residues to form highly active and selective catalysts for a wide range of chemical reactions under mild conditions. Nevertheless, many important synthetic reactions lack a naturally occurring enzymatic counterpart. Hence, the design of stable enzymes with new catalytic activities is of great practical interest, with potential applications in biotechnology, biomedicine and industrial processes. Furthermore, the computational design of new enzymes provides a stringent test of our understanding of how naturally occurring enzymes work. In the past several years, there has been exciting progress in designing new biocatalysts 2,3 .Here we describe the use of our recently developed computational enzyme design methodology 4 to create new enzyme catalysts for a reaction for which no naturally occurring enzyme exists: the Kemp elimination 5,6 . The reaction, shown in Fig. 1a, has been extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon-a process that is normally restricted by high activation-energy barriers 7,8 . Computational design methodThe first step in our protocol for designing new enzymes is to choose a catalytic mechanism and then to use quantum mechanical transition state calculations to create an idealized active site with protein functional groups positioned so as to maximize transition state stabilization (Fig. 1b). The key step for the Kemp elimination is deprotonation of a carbon by a general base. We chose two different catalytic bases for this purpose: first, the carboxyl group of an aspartate or glutamate side chain, and, second, the imidazole of a histidine positioned and polarized by the carboxyl group of an aspartate or glutamate (we refer to this combination as a His-Asp dyad). The two choices have complementary strengths and weaknesses. The advantage of the carboxylate...

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Section: Discussionmentioning

confidence: 99%

“…Rotamer pairs with a van der Waals repulsive energy less than 2.0 kcal mol 21 and hydrogen bonding energy less than 20.5 kcal mol 21 were stored in an 'interaction graph'. Matching was carried out using histidine as the catalytic residue, iterating only over histidine rotamers in the interaction graph of His-Asp and His-Glu pairs.…”

Section: Methodsmentioning

confidence: 99%

Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

1,197

1,237

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“…The reaction-based selection with designed compounds afforded improved small peptide catalysts with respect to both catalytic activity and folded state. We have demonstrated that the reaction-based selection using phage display libraries, which has been effectively used to generate larger protein catalysts (23,24,44), is useful for the development of small designer peptide catalysts. We have also demonstrated that the substrate specificity of the peptide catalyst for a small molecule substrate can be improved by a modular assembly strategy, i.e., covalent conjugation of a binding domain to the catalytic peptide module.…”

Section: Enaminone Formation and Reaction With An N-acyl--lactammentioning

confidence: 99%

Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

2005

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Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer R-helix peptide containing lysine residues. Reactionbased selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.Generation of enzyme-like small designer peptide catalysts that achieve the proficiency of natural enzymes with desired substrate specificity is a challenging task. Designer protein catalysts have been prepared by engineering naturally occurring enzymes (1-4) and by using antibody libraries (i.e., immune diversity) and selection with designed small molecule compounds (5-7). For experimental purposes, small peptides are more useful and convenient than large proteins, as long as they provide the same function, because small peptides can be more easily prepared and their characterization is simpler. However, fundamental questions remain to be answered: Can small peptides attain the catalytic efficiency of larger protein catalysts, and how can these peptide catalysts best be developed? The folded states of enzymes are key to their catalytic activity since structure can be used productively to modify the chemical reactivity of amino acid side chains. Small peptides are often limited in their potential to catalyze reactions because of the limited ability to adopt well-defined structures. Small peptides have also demonstrated limited specificity for small substrate molecules.For small catalytic peptides (<50 amino acid residues) that function in aqueous buffer, rational design has been used for the creation of catalysts of the decarboxylation of oxaloacetic acid (8-12), ester hydrolysis (13-15), and transesterifications (13). Rational design, however, is limited in throughput. A combination of rational design and librarybased methods with reaction-based selection, which has been used for the development of antibody catalysts (7), may be an attractive approach to small peptide catalyst development (16,17). Here we have explored strategies for the creation of small designer enzymes. We have developed small peptides...

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“…It is, thus, evident that innate reactivity can be deployed to capture more highly evolved biocatalysts that conserve an essential residue for covalent catalysis (12). Mechanism-based kinetic selection may, thus, be differentiated from suicide substrate selection or similar strategies that have successfully used covalent capture (13). We previously applied this approach to selection of a diversified repertoire of human Ig variable light (V L ) and variable heavy (V H ) single-chain fragments (scFv) displayed on phage particles (14).…”

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confidence: 99%

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

Смирнов

Carletti

Kurkova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. Highresolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V L ) and variable heavy (V H ) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereoand chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.catalytic antibodies | crystal structure | insecticide | organophosphate hydrolysis

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Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

Cited by 83 publications

References 44 publications

Kemp elimination catalysts by computational enzyme design

Kemp elimination catalysts by computational enzyme design

Development of Small Designer Aldolase Enzymes: Catalytic Activity, Folding, and Substrate Specificity

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

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