Mixed function oxidases steroids

Many examples of microbial hydroxylation of sterols/steroids have been reported. These hydroxylations usually involve mixed function oxidases which utilise molecular oxygen and cytochrome P-450. The reaction can be represented by ... 

Adrenodoxin. Adrenodoxin is the only iron-sulfur protein which has been isolated from mammals. This protein from mitochondria of bovine adrenal cortex was purified almost simultaneously by Kimura and Suzuki (32) and Omura et al. (33). It has a molecular weight of 12,638 (34) and the oxidized form of the protein shows maximal absorbances at 415 and 453 nm. Adrenodoxin acts as an electron carrier protein in the enzyme system required for steroid hydroxylation in adrenal mitochondria. In this system, electron transfer is involved with three proteins cytochrome P. gQ, adrenodoxin and a flavoprotein. Reduced NADP gives an electron to Tne flavoprotein which passes the electron to adrenodoxin. Finally, reduced adrenodoxin transfers the electron to cytochrome Pas shown in Fig. 3. The mechanism of cytochrome P cq interaction with steroid, oxygen and adrenodoxin in mixed-function oxidase of adrenal cortex mitochondria has been reviewed by Estabrook et al. (35). 

Megaredoxin. Another example of a bacterial mixed-function oxidase was found in the steroid 15 6-hydroxylase system of Bacillus megaterium (41). This enzyme system consists of three proteins FMN-containing flavoprotein (megaredoxin reductase), iron-sulfur protein... 

It has been well recognized that the mixed-function oxidase system of Bacillus megaterium is involved in steroid hydroxylation (, as already described above. This enzyme system is composed of a NADPH-specific FMN flavoprotein (megaredoxin reductase), an iron-sulfur protein (megaredoxin) and cytochrome P cn. The megaredoxin protein plays an important role as an intermediate component of electron transfer from reduced flavoprotein to cytochrome P en. 

Peterson RE, Imperato-McGinley, J, Gautier T, Shackleton C (1985) Male pseudohermaphroditism due to multiple defects in steroid-biosynthetic microsomal mixed-function oxidases. A new variant of congenital adrenal hyperplasia. N Engl J Med 313 1182-1191... 

Reactions described in this chapter that are catalyzed by mixed-function oxidases are those involved in fatty acyl-CoA desaturation (Fig. 21-13), leukotri-ene synthesis (Fig. 21-16), plasmalogen synthesis (Fig. 21-30), conversion of squalene to cholesterol (Fig. 21-37), and steroid hormone synthesis (Fig. 21-47). 

FIGURE 21-37 Ring closure converts linear squalene to the condensed steroid nucleus. The first step in this sequence is catalyzed by a mixed-function oxidase (a monooxygenase), for which the cosubstrate is NADPH. The product is an epoxide, which in the next step is cyclized to the steroid nucleus. The final product of these reactions in animal cells is cholesterol in other organisms, slightly different sterols are produced, as shown. 

FIGURE 21-47 Side-chain cleavage in the synthesis of steroid hormones. Cytochrome P-450 acts as electron carrier in this mixed-function oxidase system that oxidizes adjacent carbons. The process also requires the electron-transferring proteins adrenodoxin and adrenodoxin reductase. This system for cleaving side chains is found in mitochondria of the adrenal cortex, where active steroid production occurs. Pregnenolone is the precursor of all other steroid hormones (see Fig. 21-46). 

It has been elucidated by many investigators that adrenal steroid hydroxylation occurs at the 22-, 21-, 20-, 18-, 17-, and 11 -positions of the steroid molecule. All hydroxylation reactions require molecular oxygen and NAD PH, which suggests that the reaction follows the monooxygenase (mixed-function oxidase) mechanism. 

Since the major precursor of all of these hormones is cholesterol it becomes clear that, while the total biosynthetic routes are quite complex, they all involve a number of specific hydroxyla-tion reactions involving both the steroid nucleus and the side chain at C-17. Most of these are catalyzed by mixed-function oxidases involving cytochrome P-450 (Figure 1) (13-15). 

As with the mixed-function oxidases involved in xenobiotic metabolism, the substrate specificity of the steroid hydroxylases is dictated, in part, by the existence of multiple forms of both microsomal and mitochondrial cytochrome P-450s and further opportunities for specificity are provided by the distinct localization of the various enzymes in either the mitochondria or the endoplasmic reticulum. 

Most of these studies have established that the enzyme is located in the mitochondrial fraction of tissue homogenates, although the enzyme in the fat body and Malpighian tubules of Locusta is reportedly associated with the microsomal fraction (30). All studies concur that the enzyme is a cytochrome P-450-mediated mixed-function oxidase and its requirements for NADPH and 02 and sensitivity to inhibitors such as carbon monoxide, metyrapone, etc., support this conclusion. As yet, there are no reports as to whether the enzyme is associated with an iron sulfur protein similar to the adrenodoxin of the mammalian mitochondrial steroid... 

Rat liver microsomes hydroxylate 5/8-cholestane-3a ,7a,12Q -triol at C-25 and C-26 both activities are dependent on cytochrome P450 and there is some evidence that different types of the latter are involved. A mitochondrial steroid 24-hydroxylase that accepts 3a,7a,12a-trihydroxy-5/3-cholestanoic acid has been extracted from rat liver apparently this is not a mixed-function oxidase although the presence of oxygen was obligatory for its action. Bile acids hydroxylated at C-23 have been formed from sodium cholate and deoxycholate in preparations from Viperinae species and a steroid-12ct-hydroxylase from liver microsomes has been studied.Sitosterol has been confirmed to be a precursor of C24 and C29 bile acids in mammalian liver, and here hydroxylation at C-26 precedes that at C-7. ° "... 

Elepatic mixed-function oxidases are induced by many drugs, including phenytoin, phenobarbital, mitotane, aminoglutethunide, and rifampin. In addition, bp-hydroxylase and other drug-metabohzing pathways that metabolize cortisol and otlier steroids are increased by a... 

Although proteins are not cleared by cytochrome enzymes or mixed function oxidases, and would therefore not be expected to alter the pharmacokinetics of other medications, there are potential mechanisms of interaction between proteins and concomitant medications, which could affect patient exposure to the protein. For example, steroids, which alter macrophage cell trafficking (66,67), could potentially alter the clearance of large therapeutic proteins. 

Cytochrome P450 enzyme system The cytochromes P450 are mixed-function oxidases that require both NADPH and O. They are involved in a number of reactions in the conversion of lanosterol to cholesterol, as well as important steps in the synthesis of steroid hormones. Cytochromes P450 are very important in the detoxification of xenobiotics and in the metabolism of drags. 

Mixed function oxidases catalyze the oxidation of an extremely diverse array of lipophilic drug substrates as well as endobiotics such as steroid and thyroid hormones, fatty acids, and arachidonic acid metabolites. These oxidations have been broadly characterized as follows ... 

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