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F.M. Kiss et al. / FEBS Letters xxx (2015) xxx–xxx
for further functionalization, contributing to the development of
novel glucocorticoids possessing anti-inflammatory/immunomodu
latory activity with a potentially reduced risk of side effects.
Using the CYP106A1-based whole-cell system, nearly 50% of
dexamethasone was converted in 24 h, yielding four products, P1
to P4, which were all successfully isolated and characterized.
Each of the identified metabolites showed identical retention times
to the ones observed in vitro, eluting at 5.1, 6.9, 8.3 and 15.8 min,
respectively (Fig. 3B and C). The major product, P1 (37%), and P2
(6%) were identified as 15b-hydroxy- and 6b-hydroxy-derivatives
of dexamethasone, respectively. The latter one also known as the
major human metabolite of dexamethasone produced by CYP3A4
[16,30,42]. Besides the hydroxylated derivatives, two minor
11-oxidized compounds were observed and characterized as
11-ketodexamethasone (P4, 2%) and 15b-hydroxy-11-ketodexame
thasone (P3, 3%) (Scheme 2). The 11-keto compound was previ-
ously identified as a specific glucocorticoid receptor agonist,
explaining the glucocorticoid effect of dexamethasone in tissues
expressing the 11b-hydroxysteroid dehydrogenase type 2 enzyme,
responsible for 11-oxidation in mammalian tissues [34].
Fig. 4. HPLC chromatograms of the CYP106A1 catalyzed 11b-hydroxyandrostene-
The in vivo turnover of 11-OH-AD resulted in ꢀ90% conversion
within 24 h and a single main product along with traces of minor
side products, which were not observed in the in vitro system
(Fig. 4C). The major product, P1 (ꢀ10.5 min), showed identical
retention time to that of the in vitro reaction (Fig. 4B). The NMR
characterization identified P1 as the 11-keto derivative, adrenos-
terone, supporting the previously observed 11-oxidase activity of
CYP106A1 during the transformation of corticosterone and cortisol
[22] (Scheme 3). Adrenosterone is a weak androgen hormone
found only in trace amounts in mammals, but in higher quantities
in fish, where it acts as a precursor of 11-ketotestosterone, the
endogenous androgenic sex hormone [7,10]. It is sold as a dietary
supplement, reducing body fat and increasing muscle mass and
dione conversion. (A) Negative control, containing 11-OH-AD only. (B) In vitro 11-
OH-AD conversion, using bovine Adx4–108 (40
lM), bovine AdR (4 lM) and
CYP106A1 (2 M). (C) In vivo conversion of 11-OH-AD using the CYP106A1-
l
overexpressing B. megaterium MS941 strain.
and P2 were well resolved during the purification, the NMR analy-
sis of their purified fractions showed a mixture of several steroids,
containing 15b-hydroxyprednisolone as
a major compound.
Likewise, product P3, which was identified as a 2:3 mixture of
15b-hydroxyprednisone and 1,2-dihydro-15b-hydroxyprednisone
displaying 11-oxidation catalyzed by CYP106A1 (Scheme 1). The
unusual 1(2)-double bond hydrogenation has not been observed
in our previous studies [22], hence, it was proposed to be the result
of an uncharacterized enzyme present in the B. megaterium MS941
strain. It has been reported that 3-ketosteroid-dehydrogenases
present in Nocardia, Mycobacterium and Actinobacter sp. can be
responsible for both 1(2)-hydrogenase and reductase reactions
[2,17,46]. However, these enzymes have not been described so
far for B. megaterium. Therefore, the origin of the
1(2)-hydrogenase reaction remains unclear. The above described
prednisolone metabolites were previously unidentified, and
therefore, no biotechnological applications are known to date.
Nonetheless, the 15b-hydroxyl group attached to both pred-
nisolone and prednisone structures provides a new reaction site
was also proposed to be
a
selective inhibitor of
11b-hydroxysteroid dehydrogenase type I enzyme [12].
3.2. Steady state kinetic turnover in protiated and deuterated solvent
systems
The CYP106A1-mediated conversion of prednisolone and
dexamethasone resulted in multiple products, in contrast to
11-OH-AD, where only the 11-keto product formation was
detected. Thus, the latter reaction was chosen as a suitable model
for the investigation of KSIE in CYP106A1-mediated 11-oxidation.
Scheme 1. Oxidative transformation of prednisolone catalyzed by CYP106A1.
Please cite this article in press as: Kiss, F.M., et al. Identification of new substrates for the CYP106A1-mediated 11-oxidation and investigation of the reac-