176
A. Christy Hunter et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 171–176
of fungi [25] this is the first time that diol generation has been
observed with A. tamarii.
Acknowledgement
We would like to thank the University of Brighton for financial
support.
References
[1] D.R. Brannon, J. Martin, A.C. Oehischlager, N.N. Durham, L.H. Zalkow, Transfor-
mation of progesterone and related steroids by Aspergillus tamarii, J. Org. Chem.
30 (1965) 760–762.
[2] A.C. Hunter, H. Bergin-Simpson, Distinct metabolic handling of 3-hydroxy-
17a-oxa-d-homo-5␣-androstan-17-one by the filamentous fungus Aspergillus
tamarii KITA: evidence in support of steroid/hydroxylase binding hypothesis,
Biochim. Biophys. Acta 1771 (2007) 1254–1261.
Fig. 3. Transformation of C-1 and C-11 substituted metabolites with Aspergillus
tamarii KITA.
[3] A.C. Hunter, N.E. Carragher, Flexibility of the endogenous progesterone lac-
tonization pathway in Aspergillus tamarii KITA: transformation of a series of
cortical steroid analogues, J. Steroid Biochem. Mol. Biol. 87 (2003) 301–308.
[4] A.C. Hunter, S. Kennedy, S.-J. Clabby, J. Elsom, Fate of novel Quasi reverse
steroidal substrates by Aspergillus tamarii KITA: Bypass of lactonisation and
an exclusive role for the minor hydroxylation pathway, Biochim. Biophys. Acta
1734 (2005) 190–197.
[5] A.C. Hunter, J. Elsom, L. Ross, R. Barrett, Ring-B functionalized androst-4-
en-3-ones and ring-C substituted pregn-4-en-3-ones undergo differential
transformation in Aspergillus tamarii KITA: Ring-A transformation with all
C-6 substituted steroids and ring-D transformation with C-11 substituents,
Biochim. Biophys. Acta 1761 (2006) 360–366.
[6] A.C. Hunter, E. Coyle, F. Morse, C. Dedi, H.T. Dodd, S.-J. Koussoroplis, Transforma-
tion of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular
fate in lactonization and hydroxylation pathways with identification of a puta-
tive 3-hydroxy-steroid dehydrogenase/ꢀ5-ꢀ4 isomerase pathway, Biochim.
Biophys. Acta 1791 (2009) 110–117.
Incubation of the C-17 keto containing compound (7) revealed
both 1- and 11-hydroxylase activity. In order to determine if
these could act as precursor metabolites to the lactones (14) and
(15) they were isolated and subsequently incubated with A. tamarii.
Interestingly the presence of a 1-hydroyl group (12) did not inhibit
the generation of the ring-D lactone as opposed to the 11 con-
taining keto-diol (13) which did not undergo further metabolism.
This indicates that the presence of the 11-hydroxy group can
inhibit the activity of the terminal Baeyer–Villiger monooxy-
genase. In support of this the minor hydroxylation pathway
hydroxytestosterone which does not undergo further metabolism
demonstrating that the 17-hydroxy oxidase is also inhibited
[1]. This is another example in this fungus where a single func-
tional group on a steroid can have a profound effect on route of
metabolism [5]. Interestingly, it has been previously demonstrated
that the presence of an 11␣-alcohol on progesterone [5] has no
effect on the activity of either the 17-hydroxy oxidase or the
Baeyer–Villiger oxidase in this organism which, underlines the sub-
these steroids.
1-Hydroxylase activity has been previously observed in
the transformation of the 3-hydroxy-17a-oxa-d-homo-5␣-
androstan-17-one [2], an analogue of compound (9), which,
suggests that the presence of a 3␣-hydroxyl group does not
inhibit the activity of this enzyme. Furthermore in both recorded
cases the activity of this enzyme was observed post 48 h which
especially when compared to the wide range of fungi studied
to date which can perform steroidal Baeyer–Villiger oxidation
[15–20] including those of which can hydroxylate at C-11 [21–23]
(Figs. 2 and 3).
[7] E.R.H. Jones, The microbiological hydroxylation of steroids and related com-
pounds, Pure Appl. Chem. 33 (1973) 39–52.
[8] R. McCrindle, J.K. Turnbull, A.B. Anderson, Microbiological hydroxylation of
17-norkauran-16-one andent-17-norkauran-16-one with the fungus Rhizopus
nigricans, J. Chem. Soc. Perkin Trans. 13 (1975) 1202–1208.
[9] H.L. Holland, The mechanism of microbiological hydroxylation of steroids,
Chem. Soc. Rev. 11 (1982) 371–395.
[10] H.L. Holland, Models for the regiochemistry and stereochemistry of microbial
hydroxylation and sulfoxidation, Catal. Today 22 (1994) 427–440.
[11] J.R. Hanson, A.C. Hunter, The hydroxylation of steroidal ring D lactones by
Cephalosporium aphidicola, Phytochemistry 49 (1998) 2349–2353.
[12] D.N. Kirk, H.C. Toms, C. Douglas, K.A. White, A survey of the high-field 1H
NMR spectra of steroid hormones, their hydroxylated derivatives and related
compounds, J. Chem. Soc. Perkin Trans. 2 (1990) 1567–1594.
[13] J.W. Blunt, J.B. Stothers, 13C NMR of steroids—a survey and commentary, Org.
Magn. Reson. 9 (1977) 439–463.
[14] D.R. Brannon, F.W. Parrish, B.J. Wiley, L. Long, Microbiological transforma-
tion of a series of androgens with Aspergillus tamarii, J. Org. Chem. 32 (1967)
[15] H.-M. Liu, H. Li, L. Shan, J. Wu, Synthesis of steroidal lactone Penicillium citre-
oviride, Steroids 71 (2006) 931–934.
[16] A.C. Hunter, K.R. Watts, C. Dedi, H.T. Dodd, An unusual ring-A opening and other
reactions in steroid transformation by the thermophilic fungus Myceliophthora
thermophila, J. Steroid Biochem. Mol. Biol. 116 (2009) 171–177.
´
[17] T. Kołek, A. Szpineter, A. Swizdor, Baeyer–Villiger oxidation of DHEA, preg-
nenolone and androstendione by Penicillium lilacinum AM111, Steroids 73
(2008) 1441–1445.
(53%) following incubation of the 17-keto-3␣-ol (7) with the
Baeyer–Villiger monooxygenase activity observed in the first 24 h.
In comparison to a recently reported recombinant cyclohexanone
monooxygenase [24] on identical steroidal substrates (7, 8) our
whole cell system achieves a 6 times higher yield starting with
an identical substrate. Interestingly the 3␣-acetate analogue (8)
ity where Baeyer–Villiger oxidation of the C-17 ketone was first
observed at 48 h. Overall lower yields of lactones (9, 10) were
obtained following transformation of the 3␣-acetate in comparison
to the transformation of the 3␣-alcohol (7), a pattern also paralleled
with the recombinant technology [24].
´
[18] T. Kołek, A. Szpineter, A. Swizdor, Studies on Baeyer–Villiger oxidation of
steroids: DHEA and pregnenolone D-lactonization pathways in Penicillium
camemberti AM83, Steroids 74 (2009) 859–862.
[19] A.M. Khallil, M.E. Mostafa, Microbiological transformation of progesterone by
some zoosporic fungi, J. Basic Microbiol. 36 (1996) 255–259.
[20] M.A. Faramarzi, M.T. Yazdi, M. Amini, F.A. Mohseni, G. Zarrini, A. Amani, A.
Shafiee, Microbial production of testosterone and testololactone in the culture
of Aspergillus terreus, World J. Microbiol. Biotechnol. 20 (2004) 657–660.
[21] A. Bartman´ ska, J. Dmochowska-Gładysz, E. Huszcza, Steroids’transformations
in Penicillium notatum culture, Steroids 70 (2005) 193–198.
[22] M.E. Mostafa, A.A. Zohri, Progesterone side-chain degradation by some species
of Aspergillus flavus group, Folia Microbiol. 45 (2000) 243–247.
[23] A. Al-Aboudi, M.Y. Mohammad, S.G. Musharraf, M.I. Choudhary, Atta-ur-
Rahman, Microbial transformation of testosterone by Rhizopus stolonifer and
Fusarium lini, Nat. Prod. Res. 22 (2008) 1498–1509.
[24] E. Beneventi, G. Ottolina, G. Carrea, W. Panzeri, G. Fronza, P.C.K. Lau, Enzymatic
Baeyer–Villiger oxidation of steroids with cyclopentadecanone monooxyge-
nase, J. Mol. Catal. B: Enzym. 58 (2009) 164–168.
[25] M.V. Donova, O.V. Egorova, V.M. Nikolayeva, Steroid 17-reduction by
microorganisms-a review, Process Biochem. 40 (2005) 2253–2262.
Aside from oxidation, a minor reduction pathway was observed
with transformation of the C-17 ketone of compound (7) to a C-
17-alcohol. Although this reduction is common in a wide range