Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry.
Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.
Lisurek
Michael
M
Universität des Saarlandes, FR 8.8-Biochemie, Postfach 151150, 66041 Saarbrücken, Germany.
Kang
Min-Jung
MJ
Hartmann
Rolf W
RW
Bernhardt
Rita
R
D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D011374:Progesterone / Q000096:biosynthesis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System