• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1750-1759
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• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• Toxicological Research
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• v.13 no.1_2
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• pp.117-125
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• 1997

In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$-aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43$\pm$0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32$\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.