Cytochrome P450 hydroxylases

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein FejSj. Carbon monoxide (CO) inhibits the indicated step.

MBC, BP3 presents hydroxylated groups that allow conjugation in the absence of cytochrome P450 hydroxylase activity. 

The precursor, 7-dehydrocholesterol is converted by a non-enzymatic reaction to cholecalciferol (calciol). This reaction occurs in skin exposed to sunlight due to irradiation by UV-B light at a wavelength of about 300 nm. Cholecalciferol is transported via carrier proteins to the liver where hydroxylation at carbon-25 occurs in a reaction catalysed by a microsomal cytochrome P450 hydroxylase to form calcidiol. This compound travels to the kidney attached to specific binding proteins, where another cytochrome P450 enzyme, mitochondrial 1-a-hydroxylase, introduces a second hydroxyl group in to the molecule to form the active calcitriol. 

Within the adrenal cortex (the outer portion of the adrenal glands) progesterone is converted into two groups of hormones of which cortisol and aldosterone are representative.263 Two different cytochrome P450 hydroxylases, found in the ER and specific for C-21 and C-17a, respectively, together with a mitochondrial cytochrome P450 specific for C-lip (Eq. 18-55) participate in formation of cortisol.264 Two of the same enzymes together with additional hydroxylases are required to form aldosterone. 

Basch J, Chiang S-H. Cloning and expression of a cytochrome P450 hydroxylase gene from Amycolatopsis orientalis hydroxylation of epothilone B for the production of epothilone F. Journal of Industrial Microbiology and Biotechnology 34(2), 171, 2007. 

Then, the amino acid precursor 105 is selected by the adenylation domain A and attached to the peptidyl carrier protein PCP of the SalB didomain. PCP-bound 105 is subsequently oxidized by the cytochrome P450 hydroxylase SalD. Fusion of the PKS-and the NRPS-derived precursors by the C-terminal condensation domain of SalA, leads to a PCP-bound linear intermediate, which after bicyclization and concurrent release from the synthetase yields the fully assembled salinosporamide molecules by an unknown process. The observed structural diversity of the salinosporamide family is essentially due to the relaxed substrate specificity of the AT domains of SalA. Although selection of an acetate starter unit by ATL leads to the typical methyl substituent at C-3 of the salinosporamides, an alternative priming with propionate would produce the C-3-ethyl derivative salinosporamide 194. The promiscuity of ATj in turn facilitates the formation of the observed variability in the substitution pattern at C-2. For the assembly of 86, ATj incorporates the halogenated PKS extender unit chloroethylmalonyl-CoA, which is unique to the salinosporamide family. 

On the way to baccatin III, there are several more steps. Two oxygenation reactions remain to be defined, and the corresponding gene are missing. These are the presumed cytochrome P450-mediated taxoid C-ip-hydroxylase and the C-4p,C-20-epoxidase, leading to the oxetane formation the oxidation of the C-9a-hydroxyl function, likely mediated by a cytochrome P450 hydroxylase and the transfer of three ester functions C-2 benzoate, C-4 acetate, and C-10 acetate. 

The coupling of dopamine and 3,4-DHPAA permits bypassing cytochrome P450 hydroxylase (CYP80B). Moreover, deamination catalyzed by microbial MAO means that reticuline can be obtained from a single substrate dopamine. The biosynthetic pathway of reticuline from L-tyrosine in plants requires nine enzymes. However, the shortcut biosynthetic pathway from dopamine in microbial production requires only five enzymes. 

One of the most commonly found mid-chain hydroxylated components is dihydroxyhexadecanoic acid, which has hydroxyl moieties at C-10, C-9, C-8 or C-7, and on C-16 (232, 243, 244). A crude cell-free preparation from excised epidermis of V. faba catalyzed C-10 hydroxylation of 16-hydroxyhexadecanoic acid (473). This hydroxylation reaction was also catalyzed by the endoplasmic reticulum fraction from the embryonic shoots of K faba. This preparation required O2 and NADPH to catalyze mid-chain hydroxylation and the activity was inhibited by the typical mixed-function oxidase inhibitors and also by CO (427). The inhibition by CO was photoreversible, as expected of a cytochrome P450 hydroxylase. 

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