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@ARTICLE{Dick:818274,
author = {Dick, Markus and Hartmann, Rudolf and Weiergräber, Oliver
H. and Bisterfeld, Carolin and Classen, Thomas and
Schwarten, Melanie and Neudecker, Philipp and Willbold,
Dieter and Pietruszka, Jörg},
title = {{M}echanism-based inhibition of an aldolase at high
concentrations of its natural substrate acetaldehyde:
structural insights and protective strategies},
journal = {Chemical science},
volume = {7},
number = {7},
issn = {2041-6539},
address = {Cambridge},
publisher = {RSC},
reportid = {FZJ-2016-04748},
pages = {4492 - 4502},
year = {2016},
abstract = {2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is used in
organic synthesis for the enantioselective reaction between
acetaldehyde and a broad range of other aldehydes as
acceptor molecules. Nevertheless, its application is
hampered by a poor tolerance towards high concentrations of
acetaldehyde, its natural substrate. While numerous studies
have been performed searching for new, more
acetaldehyde-resistant DERAs, the mechanism underlying this
deactivation process has remained elusive. By using NMR
spectroscopy on both the protein and the small-molecule
scale, we could show that a reaction product binds to the
inner part of the enzyme, and that this effect can be partly
reversed via heating. The crystal structure of DERA before
and after acetaldehyde incubation was determined at high
resolution, revealing a covalently bound reaction product
bridging the catalytically active lysine (K167) to a nearby
cysteine (C47) in the deactivated enzyme. A reaction
mechanism is proposed where crotonaldehyde as the aldol
product of two acetaldehyde molecules after water
elimination forms a Schiff base with the lysine side chain,
followed by Michael addition of the cysteine thiol group to
the Cβ atom of the inhibitor. In support of this mechanism,
direct incubation of DERA with crotonaldehyde results in a
more than 100-fold stronger inhibition, compared to
acetaldehyde, whereas mutation of C47 gives rise to a fully
acetaldehyde-resistant DERA. Thus this variant appears
perfectly suited for synthetic applications. A similar
diagnostic and preventive strategy should be applicable to
other biocatalysts suffering from mechanism-based inhibition
by a reactive substrate, a condition that may be more common
than currently appreciated in biotechnology.},
cin = {ICS-6 / IBOC / IBG-1},
ddc = {540},
cid = {I:(DE-Juel1)ICS-6-20110106 / I:(DE-Juel1)IBOC-20090406 /
I:(DE-Juel1)IBG-1-20101118},
pnm = {553 - Physical Basis of Diseases (POF3-553) / 581 -
Biotechnology (POF3-581)},
pid = {G:(DE-HGF)POF3-553 / G:(DE-HGF)POF3-581},
typ = {PUB:(DE-HGF)16},
UT = {WOS:000378715000066},
pubmed = {pmid:30155096},
doi = {10.1039/C5SC04574F},
url = {https://juser.fz-juelich.de/record/818274},
}