Metabolism of steroidal lactones by the fungus Corynespora cassiicola CBS 161.60 results in a mechanistically unique intramolecular ring-D cyclization resulting in C-14 spiro-lactones.

The fungus Corynespora cassiicola metabolises exogenous steroids in a unique and highly specific manner. Central to this, is the ability of this organism to functionalise substrates (androgens, progestogens) at the highly stereochemically hindered 8ß-position of the steroid nucleus. A recent study has identified that 8ß-hydroxylation occurs through inverted binding in a 9α-hydroxylase. In order to discern the metabolic fate of more symmetrical molecules, we have investigated the metabolism of a range of steroidal analogues functionalised with ring-D lactones, but differing in their functional group stereochemistry at carbon-3. Remarkably, the 3α-functionalised steroidal lactones underwent a mechanistically unique two step intramolecular cyclisation resulting in the generation of a ring-D spiro-carbolactone. This rapid rearrangement initiated with hydroxylation at carbon 14 followed by transesterification, resulting in ring contraction with formation of a butyrolactone at carbon-14. Remarkably this rearrangement was found to be highly dependent on the stereochemistry at carbon-3, with the ß-analogues only undergoing 9α-hydroxylation. The implications of these findings and their mechanistic bases are discussed.

Metabolic fate of 3α,5-cycloandrostanes in the endogenous lactonization pathway of Aspergillus tamarii KITA.

A series of 3α,5-cycloandrostane analogues with a range of functionality (6α and 6ß alcohols and ketone) at carbon 6 were tested in the endogenous lactonization pathway in Aspergillus tamarii KITA. This metabolic route converts progesterone to testololactone in high yield through a four step enzymatic pathway. To date, no studies have looked at the effect of steroids devoid of polar functionality at carbon 3 and their subsequent metabolic fate by fungi which contain Baeyer-Villiger monooxygenases. Incubation of all of the cycloandrostane analogues resulted in lactonization of ring-D irrespective of C-6 stereochemistry or absence of C-3 functionality. Presence of 6ß-hydroxy group and the C-17 ketone was required in order for these analogues to undergo hydroxylation at C-15ß position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data.

Transformation of structurally diverse steroidal analogues by the fungus Corynespora cassiicola CBS 161.60 results in generation of 8ß-monohydroxylated metabolites with evidence in favour of 8ß-hydroxylation through inverted binding in the 9α-hydroxylase.

Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8ß-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8ß-position. The collective data has demonstrated the first time that 8ß-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8ß and 15ß has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8ß-hydroxylation of these steroids is discussed.

Transformation of a series of saturated isomeric steroidal diols by Aspergillus tamarii KITA reveals a precise stereochemical requirement for entrance into the lactonization pathway.

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17ß-diol which, uniquely underwent oxidation of the 17ß-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17ß-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11ß. Only 5α-androstan-3ß,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6ß and 7ß positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement.

Transformation of some 3alpha-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway.

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17beta-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11beta-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3beta-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3alpha-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11beta-position (normal binding), indicating that the 3alpha-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1beta-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11beta-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11beta-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3alpha-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3alpha-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17beta-alcohol for the first time in this organism.

An unusual ring--a opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila.

A series of steroids (progesterone, testosterone acetate, 17beta-acetoxy-5 alpha-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17beta side-chain. This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17beta-acetoxy-5 alpha-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17beta-alcohol of testosterone. All steroidal metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3beta-hydroxy-steroid dehydrogenase/Delta5-Delta4 isomerase pathway.

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3beta-hydroxy-steroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3beta-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3beta-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13alpha-methyl analogue. In contrast to previous studies of fungi with 3beta-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one were metabolized solely through the putative 3beta-HSD/isomerase pathway, indicating that a 17beta-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3beta-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.

Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312.

This paper demonstrates for the first time transformation of a series of steroids (progesterone, androst-4-en-3,17-dione, testosterone, pregnenolone and dehydroepiandrosterone) by the thermophilic fungus Rhizomucor tauricus. All transformations were found to be oxidative (monohydroxylation and dihydroxylation) with allylic hydroxylation the predominant route of attack functionalizing the steroidal skeleta. Timed experiments demonstrated that dihydroxylation of progesterone, androst-4-en-3,17-dione and pregnenolone all initiated with hydroxylation on ring-B followed by attack on ring-C. Similar patterns of steroidal transformation to those observed with R. tauricus have been observed with some species of thermophilic Bacilli and mesophilic fungi. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The application of thermophilic fungi to steroid transformation may represent a potentially rich source for the generation of new steroidal compounds as well as for uncovering inter and intraspecies similarities and differences in steroid metabolism.