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Adrenodoxin Reductase (adrenodoxin + reductase)
Selected AbstractsCovalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450sccFEBS JOURNAL, Issue 6 2001Edman degradation of complexes of the steroidogenic hydroxylase system, Mass spectrometry NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin ,P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin,P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2CHEMBIOCHEM, Issue 5 2010Daniela Zehentgruber Dr. Abstract Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate CH bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g,L,1,d,1. Product extraction with absorber resins allowed the recovery of quantitative amounts of 15,-OH-progesterone and 15,-OH-testosterone and also the reuse of the biocatalyst. [source] |