Application of Modern Enzyme Design - ETH E

Diss. ETH No. 21428
Application of Modern Enzyme Design
Technologies and Strategies
for their Refinement
A dissertation submitted to
ETH Zurich
for the degree of
Doctor of Sciences
presented by
NATHALIE PREISWERK
M.Sc. in Chemistry, EPF Lausanne
born on January 23rd, 1984
citizen of Switzerland and Bolivia
Accepted on the recommendation of
Prof. Dr. D. Hilvert, examiner
Prof. Dr. D. Neri, co-examiner
Zürich, 2013

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ABSTRACT
Enzymes are the most powerful catalysts. Their use as alternatives to
small molecule and metal-based catalysts for industrial processes is very
appealing but often limited by the narrow range of substrates they convert and
their limited stability in industrial settings. Scientists have therefore developed
strategies to generate artificial enzymes with tailored properties. This thesis
describes the study of three artificial enzymes.
In order to mimic natural enzymes efficiently, a precise understanding of
the processes responsible for their remarkable activities is necessary. The
cotranslational incorporation of selenocysteine in natural proteins is extremely
costly and requires the expression of complex machineries. The higher
nucleophilicity of selenium is often claimed to justify its insertion into proteins.
In Chapter 2, we compare the reductive activity of an engineered dithiol oxidase
DsbA variant in which the catalytic cysteine at position 30 was replaced by
selenocysteine with that of its cysteine-containing counterpart. This system
allows unbiased comparison of nucleophilicity between selenocysteine and
cysteine since, in this protein environment, both side chains are in their
deprotonated state under physiological conditions. We show that selenocysteine
provides a 10-fold catalytic advantage over cysteine for the reduction of a
variety of oxidants such as hydroperoxides, disulfides and diselenides. Although
a 10-fold difference in nucleophilicity between selenols and thiols seems rather
modest to justify the energy-demanding selenocysteine incorporation into
proteins, its combination with the lower pKa of selenols which increases the

Abstract
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concentration of deprotonated reactive species, together with the novel redox
properties of the 21st proteinogenic amino acid, under physiological conditions
apparently make this energy expense worthwhile.
One enticing goal in enzyme design is the generation of proteins with
catalytic activities unknown to Nature. In Chapter 3, we describe the
optimization by directed evolution of a computationally designed
Diels-Alderase that catalyzes the non-biological cycloaddition of
4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-
dimethylacrylamide. In order to evolve this Diels-Alderase, we have developed
a medium throughput tandem-mass spectrometry screening assay consisting of
three consecutive steps. First, the desired reaction is carried out in cell lysate.
The product of the reaction is then extracted in a solvent compatible with
electrospray ionization (ESI) and finally analyzed without further work-up by
tandem mass spectrometry. By applying this assay to the laboratory evolution of
the initial computational design and of a computationally refined version of the
design, we have generated DA CE20, a Diels-Alderase variant with an effective
molarity (kcat/kuncat) 100-fold higher than that of the initial computational design.
The evolved DA CE20 is 100-fold more efficient at catalyzing the target Diels-
Alder reaction than the catalytic antibodies generated for the same reaction and
has the same levels of activity than the best biocatalysts ever generated for any
Diels-Alder reaction. Structural characterization of DA CE20 in complex with a
product analog has allowed us to confirm that the catalytic machinery of the
enzyme, consisting of two residues, a tyrosine and a glutamine, that interact
with the diene and dienophile, respectively, is very similar to what was intended
by design. Nevertheless, the catalytic activity of DA CE20 remains modest
compared to other evolved computational designs and natural enzymes. The
remarkable rigidity of its scaffold may have limited the extent to which it could
be improved. It is also possible that the catalytic mechanism based on hydrogen

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bond interactions chosen during computational design is not optimal. Future
investigation of other protein scaffolds and alternative mechanisms, particularly
metal ion catalysis, may yield highly efficient Diels-Alder biocatalysts.
An alternative approach to the design of enzymes with novel activities
relies on redesigning the substrate specificity of an existing enzyme while
retaining its catalytic machinery. Applying this strategy to computationally
redesign the substrate specificity of human guanine deaminase towards
ammelide has yielded hGDA-des, a protein that hydrolyzes the new substrate,
albeit seven orders of magnitude less efficiently than the native activity of
wild-type guanine deaminase towards guanine. In Chapter 4, we describe our
efforts to develop a medium-throughput screening assay for directed evolution
of hGDA-des. We first attempted to adapt the mass spectrometry assay
described in Chapter 3 but the strong similarities between the substrate,
ammelide, and the product, cyanuric acid, in terms of solubility and molecular
weight rendered the implementation of this mass spectrometry assay
problematic. We therefore explored the utility of a glutamate dehydrogenase-
coupled colorimetric assay for detecting the ammonium ions that are produced
upon ammelide deamination. Glutamate dehydrogenase catalyzes the reductive
amination of α‐ketoglutarate to glutamate in the presence of ammonium while
simultaneously oxidizing NADH. Cofactor oxidation is easily monitored by a
decrease in absorbance at 340 nm. Although the developed medium-throughput
method is not yet fully optimal, it was successfully used to probe the validity of
the computational approach. We found that while computational design
succeeded at remodeling the flexible loop of guanine deaminase to recognize
ammelide, it failed at predicting the orientation of one crucial side-chain with
the atomic-level precision needed for high catalytic efficiency. Future
improvements in the screening method and its application to the evolution of
hGDA-des are expected to yield considerable improvements in activity and

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provide concrete suggestions for the refinement of the current computational
approaches.
In summary, this thesis has contributed clues for the refinement of several
current strategies for enzyme design. Rational introduction of novel
functionalities in enzymes will extend the range of transformations they
catalyze. Iterations of computational design with ever-more sophisticated
algorithms followed by directed evolution is a promising approach for the
generation of highly efficient enzymes in the future.