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@PHDTHESIS{Bracco:444861,
author = {Bracco, Paula},
othercontributors = {Schallmey, Anett},
title = {{S}elective steroid hydroxylation by bacterial cytochrome
{P}450 monooxygenases},
address = {Aachen},
reportid = {RWTH-CONV-145180},
pages = {VII, 263 S. : Ill., graph. Darst.},
year = {2014},
note = {Aachen, Techn. Hochsch., Diss., 2014},
abstract = {Regio- and stereoselective hydroxylation of steroid
molecules remains a challenge in industrial steroid hormone
synthesis. Here, the use of cytochrome P450 monooxygenases
is of high interest due to their remarkable ability to
catalyze the direct hydroxylation of non-activated carbon
atoms in a regio- and stereoselective manner. This allows
also the selective oxyfunctionalization of steroids, thus
avoiding the use of protecting groups and several
time-consuming chemical steps. In this thesis, the use of
bacterial P450s, especially CYP154C5 from Nocardia
farcinica, was investigated for the selective hydroxylation
of various steroids. In particular, an efficient whole-cell
biocatalyst was developed based on the recombinant
expression of CYP154C5 in Escherichia coli together with
putidaredoxin and putidaredoxin reductase from Pseudomonas
putida for electron transfer. Cofactor regeneration was
achieved by the simple addition of glucose and with the help
of hydroxypropyl-beta-cyclodextrin for substrate
solubilization, several steroid molecules could be
successfully converted with up to 15 mM initial steroid
concentration using this whole-cell system. Additionally,
16alpha-hydroxylated steroids, which are important
precursors for the synthesis of highly potent
glucocorticoids, were selectively produced on preparative
scale with total turnover numbers (TTN, µmol substrate
consumed µmol-1 CYP154C5) exceeding 2000 and space-time
yields of several grams per liter a day. Furthermore,
CYP154C5´s crystal structure with six different steroid
substrates bound in the active site was determined to
identify key residues in the active site that are involved
in substrate binding and therefore responsible for the
remarkable selectivity of the P450 monooxygenase. Among the
21 residues forming the active site, four were suspected to
play an important role in substrate binding and catalytic
activity. In particular, residues M84 and F92 were
identified to be involved in hydrophobic interactions with
the steroid core, whereas residues Q239 and Q398 were found
to form hydrogen bonds with oxyfunctional groups at
positions C3 and C17 of the steroid substrate. Two
strategies were applied to further investigate the
selectivity of CYP154C5. Firstly, the four mentioned
residues were exchanged by alanine through site-directed
mutagenesis in order to remove the mentioned
enzyme-substrate interactions. The resulting mutants were
analyzed in the conversion of six steroid substrates by
determining dissociation constants (KD), turnover numbers
(TON), TTN and coupling efficiencies using purified
proteins. Results confirmed the importance of the four
residues for substrate binding and conversions as indicated
by the decreased substrate affinity and overall efficiency
of the conversion. As the only exception, nandrolone was
found to be better converted by mutant Q239A as compared to
wild-type CYP154C5. Interestingly, with a single mutation in
the active site a secondary hydroxylation product was
obtained in the conversion of progesterone by CYP154C5-F92A,
thus changing the regioselectivity of this enzyme. In a
second approach, CYP154C5 wild type was tested in the
conversion of selected steroids lacking key functional
groups in their structure that could substantially affect
substrate binding and therefore also selectivity of the
enzyme. Interestingly, here the regioselectivity of CYP154C5
was altered when a steroid lacking a functional group at
position C17 was used as substrate as indicated by the
formation of the 15alpha- instead of 16-hydroxylated
product. Furthermore, the use of a steroid substrate bearing
no functional group at C3 also resulted in a changed
regioselectivity of CYP154C5 as here four different
hydroxylation products were obtained. In contrast, steroid
substrates with large substituents at position C17 were not
converted by the P450. These findings shed light on the
possibility to create a CYP154C5-based tool box for
selective steroid hydroxylation by protein engineering.
Interestingly, a much smaller substrate, beta-ionone, was
also selectively monohydroxylated by this enzyme. CYPs from
other bacterial sources were also tested in steroid
conversions within this thesis. As novelty, we could show
for the first time that also 3beta-hydroxy-Delta5-steroids
are hydroxylated by CYP106A2 from Bacillus megaterium in a
regioselective manner. Furthermore, CYP110D1 from Nostoc sp.
was shown for the first time to hydroxylate also other
steroid molecules than testosterone. In summary,
industrially important target positions for steroid
hydroxylation such as 7alpha-, 7beta-, 11-, 15alpha- and
16alpha- were successfully hydroxylated within this thesis
by the use of different bacterial P450 enzymes and the
enzymes’ selectivities varied depending of the applied
substrates.},
keywords = {Cytochrom P-450 (SWD) / Regioselektivität (SWD) /
Stereoselektivität (SWD)},
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-opus-50936},
url = {https://publications.rwth-aachen.de/record/444861},
}
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