DUGiDocsComputational exploration and design of HHDH variants with novel synthetically useful functionalities
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Enzymes are the best catalysts. They are the main catalysts in
cells and have been exposed to millions of years of natural evolution
by including random mutations in their sequence and posterior
selection. Some enzymes show extreme catalytic rates, selectivity,
or stability, but not all are suitable for industrial or pharmaceutical
applications. Their use in industrial contents would be very advantageous,
thanks to the fact that enzymes are naturally biodegradable
molecules, work in water-based solvents, and are non-toxic. These
properties are critical for a suitable future for the new generations.
It is crucial to design enzymes for catalyzing industrially relevant
reactions.
One enzyme family with significant usage in the pharmaceutical
industry is Halohydrin Dehalogenasses (HHDH). These enzymes
convert (S)-4-chloro-3-hydroxybutyrate into ethyl (R)-4-cyano-
3-hydroxybutyrate, a precursor of statin drugs that need to be
enantiomerically pure. Not all HHDHs can perform this catalysis
due to their inability to accept the substrate (they have a limited
substrate scope), and insufficient enantioselectivity, stability, or
activity. The design of new enzymes that display good properties in
the selected industrial environment is nowadays possible, thanks to
the experimental Directed Evolution technique. Still, this protocol
mutates residues randomly. The effect of the mutations is not
rationalized and usually requires the production and screening of
multiple (thousands) variants, which has a high cost associated.
Computational protocols, based, for instance, on Molecular Dynamics
(MD) simulations, allow for rationalizing the effect of the
introduced mutations onto the ensemble of conformations that the
enzymes can explore. Still, analyzing MD simulation outputs can be
challenging, and there is no gold standard that gives good results
and is computationally feasible. From these MD simulations, the
identification of which amino acid positions need to be changed to
enhance a given property is also not straightforward.
In this thesis, a novel pipeline for analyzing the variance obtained
during the MD simulations and the accessible tunnels has
been developed and is described in Chapter 4. This new protocol
was applied to explore the tunnels in all HHDH families, compare
the results with the reported features of each HHDH, and propose
new mutagenesis sites (Chapter 5). Chapter 6 describes the
newly discovered D-family HHDHs and some proposed variants
from Prof. Anett Schallmey’s group, and the thermal stability mechanism
is unveiled. Finally, in Chapter 7, the most evolved variant
generated via Directed Evolution, i.e., HheC R18, is experimentally
and computationally characterized to rationalize the effect of
the randomly introduced mutations and how these affect the stability,
oligomerization, cooperativity, and catalytic parameters of the
HHDH enzyme.
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