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@PHDTHESIS{Mertens:567406,
author = {Mertens, Alan},
othercontributors = {Schallmey, Anett and Schwaneberg, Ulrich},
title = {{U}ntersuchung verschiedener {E}in- und
{D}reikomponenten-{P}450-{S}ysteme für die {A}nwendung in
der {B}iokatalyse},
school = {RWTH Aachen},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2016-00818},
pages = {1 Online-Ressource (VII, 262 Seite) : Diagramme},
year = {2015},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2016; Dissertation, RWTH Aachen, 2015},
abstract = {Cytochrome P450 monooxygenases are enzymes of high interest
which are applied frequently in biocatalysis because they
are able to catalyze hydroxylation reactions and many other
reactions. As these enzymes need electrons for catalysis,
cytochrome P450 monooxygenases can be grouped into different
classes according to the system used for electron transfer.
P450 enzymes which belong to three-component systems require
redox proteins like ferredoxins and ferredoxin reductases
while one-component P450 monooxygenases are
redox-self-sufficient and therefore do not depend on
additional redox proteins.The first project of this thesis
dealt with the investigation of putative electron transfer
proteins from the thermophilic bacterium
$\textit{Thermobifida fusca}$ in order to identify the
native redox partners of CYP154H1, a thermostable
three-component P450 monooxygenase. Furthermore,
thermostable redox partners for their application in
biocatalysis with other three-component P450 monooxygenases
should be identified. In the first step, selected redox
proteins from $\textit{T. fusca}$ (five ferredoxins, one
flavodoxin and four ferredoxin reductases) were
recombinantly expressed in $\textit{E. coli}$. After
activity determination, a number of redox protein
combinations were applied in biocatalysis experiments with
several cytochrome P450 monooxygenases (CYP154H1, CYP154C5
from $\textit{Nocardia farcinica}$ and CYP106A2 from
$\textit{Bacillus megaterium}$). The data analysis revealed
that none of the tested combinations resulted in a
conversion which differed significantly from the conversion
in the respective negative controls. Under the premise that
the natural redox partners for CYP154H1 would have been
active and correctly folded, these findings indicate that no
natural redox partners were among the tested electron
transfer proteins. In addition, the different redox proteins
were obviously not capable to transfer electrons to CYP154C5
and CYP106A2 under the applied conditions.The second project
of this thesis comprised the investigation of the
self-sufficient cytochrome P450 monooxygenases CYP102K1 from
the gram-negative bacterium $\textit{Azorhizobium
caulinodans}$ and CYP102$_{Nmu}$ from the gram-positive
bacterium $\textit{Nakamurella multipartia}$. The heme
domain of these enzymes and the heme domain of CYP102A1 (a
well-characterized self-sufficient P450) share a sequence
identity of less than 40 \%. Hence one goal of this project
was to investigate whether such low sequence identities are
accompanied by different substrate spectra. It was possible
to express soluble CYP102K1 recombinantly in $\textit{E.
coli}$ while all attempts to achieve soluble expression of
CYP102$_{Nmu}$ were not successful. In addition to the
full-length protein of CYP102K1, also an N-terminally
truncated variant (N‘-short-CYP102K1) was generated by
removing the 80 amino acid long tail at the N-terminus of
CYP102K1, which is not present in CYP102A1. Both CYP102K1
variants were produced and purified in sufficient yields and
NADPH was found to be the preferred cofactor in both cases.
Investigations of the substrate spectrum revealed that
linear saturated fatty acids with a chain length of ten to
fifteen C-atoms represent suitable substrates for the
CYP102K1 variants. The product analysis disclosed a distinct
regioselectivity of the CYP102K1 variants towards the ω-1-
to ω-3-position. Interestingly, this shows that CYP102K1
and CYP102A1 convert similar substrates with comparable
regioselectivity despite their low sequence identity. In
order to extend the substrate spectrum of CYP102K1, two
different dicarboxylic acid monoesters (pimelic acid
monopentylester and succinic acid monooctylester) were
synthesized in preparative scale using the lipase CalB.
These esters were applied as potential substrates in
bioconversion experiments using CYP102K1. Indeed for both
compounds product formation was detected although the
conversion was rather low. Finally, it can be concluded that
with the characterization of CYP102K1 for the first time the
experimental investigation of a self-sufficient CYP102
enzyme from a gram-negative bacterium was successful.},
cin = {160000 / 162610},
ddc = {570},
cid = {$I:(DE-82)160000_20140620$ / $I:(DE-82)162610_20140620$},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-rwth-2016-008182},
url = {https://publications.rwth-aachen.de/record/567406},
}
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