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.
Christy Hunter
A
A
Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, East Sussex BN2 4GJ, UK. c.hunter@bton.ac.uk <c.hunter@bton.ac.uk>
Khuenl-Brady
Hedda
H
Barrett
Patrice
P
Dodd
Howard T
HT
Dedi
Cinzia
C
D000732:Androstanols / Q000032:analysis / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D055162:Biocatalysis; D001711:Biotransformation / Q000502:physiology; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013055:Spectrophotometry, Infrared; D013237:Stereoisomerism; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013379:Substrate Specificity
D000732:Androstanols; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase