Steroidal hydroxylation

The oxidizing properties of positive halogen precursors have been reviewed by Filler. Compounds which have been used in steroid hydroxyl oxidations are Wbromosuccinimide Wbromoacetamide (NBA), A-bro-... 

The reactivity of various steroid alcohols decreases in the order primary > secondary (equatorial) > secondary (axial) > tertiary. The only systematic investigation relating to the selective protection of steroidal hydroxyl functions has been carried out with the cathylate (ethyl carbonate) group. Since only equatorial hydroxyl groups form cathylates this ester has been used as a diagnostic tool to elucidate the configuration of secondary alcohols. 

Table 9.7 Examples of steroid hydroxylation using call-free extracts. (Data cited by Martin CKA, Steroids in Biotechnology Vol 6a. Edited by Kieslich K 1984 Verlag Chemie, Weinheim).

Harlow, G.R. and Halpert, J.R. (1998) Analysis of human cytochrome P450 3A4 cooperativity construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. Proceedings of the National Academy of Sciences of the United States of America, 95 (12), 6636-6641. 

Figure 2.2 Regio- and stereo-selective steroid hydroxylation...

A screening of about 40 strains currently used for steroid hydroxylation was undertaken, using reverse-phase HPLC and UV absorption to detect and quantitate metabolite formation. Most of the strains tested were able to metabolize extensively this substrate within a 1- to 5-day period, producing in the incubation medium variable amounts of at least eight hydroxylated metabolites (80-87), essentially depending on the strain used [189]. 

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). 

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. 

Several hydroxylation biotransformations have been conunercialised, such as steroid hydroxylation, e.g. the 11-bf-hydroxylation of progesterone by Rhizopus arrhizus, 6-... 

Schwab GE, Raucy JL, Johnson EF. Modulation of rabbit and human hepatic cytochrome P-450-catalyzed steroid hydroxylations by alpha-naphthoflavone. Mol Pharmacol 1988 33(5) 493 199. 

Iron-sulfur proteins contain non-heme iron and inorganic (acid-labile) sulfur in their active centers as 4Fe-4S or 2Fe-2S or, in the case of rubredoxin, as one iron alone. The iron is always bonded to cysteine sulfur. They catalyze redox reactions between +350 and —600 mV (hydrogen electrode = —420 mV). They are usually of low molecular weight (6000-15,000 Daltons) but can form complex enzymes with molybdenum and flavin. They occur as soluble or membrane-bound proteins and catalyze key reactions in photosynthesis, oxidative phosphorylation, nitrogen fixation, H2 metabolism, steroid hydroxylation, carbon and sulfur metabolism, etc. They occur in all organisms so far investigated and may... 

Excellent reviews in this field have recently appeared (12, 34) and general discussions of the biological reactions and chemical nature of these electron-transferring non-heme iron proteins were held at recent symposia (56, 71). This paper is intended to be a review of recent works in our laboratory pertaining to non-heme iron protein serving as an electron transfer intermediate in steroid hydroxylation in mammalian glands. 

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. 

The same electron transfer system operates in the 22-, 20-, and 18-hydroxylations as well as in the 11 -hydroxylation. Further, the identical electron transfer system appears to be pertinent to the system of steroid hydroxylation in testis and ovary. 22-, 20-, 18-, and 11 p-hydroxy-lases are all distributed in the mitochondrial fraction of adrenal cortex, whereas 21- and 17-hydroxylases are located in the microsomal fraction. The electron transfer system in the microsomal steroid hydroxylation has not yet been established. 

Our study on the distribution of electron transferring proteins in animal sources is still in progress. From present knowledge, adrenodoxin can be found in adrenal cortexes from pig, beef, and rat. Further, a similar protein was isolated from pig testis (see II-A-2), and it was also found in the ovary. However, brain, heart, liver, kidney, and pancreas appear to lack adrenodoxin-like protein. If this is correct, the proteins of the ferredoxin family are located solely in the glands which directly act in the biosynthesis of steroid hormones. It is of interest that adrenodoxin-like protein does not participate in the steroid hydroxylation involved in cholesterol and cholic acid biosyntheses. All of these reactions without the participation of adrenodoxin are similar to enzymes responsible for microsomal non-specific hydroxylation, which consist of the following sequence of electron transfer ... 

Testis iron protein steroid hydroxylation (25, 43) active... 

Ring-c aromatic steroids hydroxylated at C-7 have also been described.49 Direct dehydrogenation of 3/3 -hydroxy-17,17-dimethyl-18-nor-5 a -androst-13-ene with... 

The enzyme involved in the reaction is a heme-containing cytochrome P-450. Several relatives of it are involved in a large number of steroids hydroxylations and, overall, play an important role in steroidogenesis. 

Steroid-metabolizing mitochondria, e.g., those from beef adrenal cortex (76, 82-84) and porcine corpus luteum (86, 86), were recently shown to contain an active energy-linked transhydrogenase that appears to play a role in steroid hydroxylation reactions (see Section IV). Under proper assay conditions all mitochondrial transhydrogenases hitherto described may be coupled to respiratory energy or ATP. 

Beauveria suffiirescens (ATCC7159) Fungi imperfect 1 la-Hydroxylation of steroids, hydroxylation of cyclic and acyclic amides, tricyclic terpenes and aromatics... 

Calonectria decora (ATCC 14767, NRRL 2380) Ascomycetes Steroid hydroxylation and dehydrogenadon, hydroxylation of cyclic hydro ub(ms... 

Cunninghamella elegans (NRRL 1393) Phycomycetes Steroid hydroxylation and allylic hydroxylation... 

Multiple hydioxylation is one of the most frequently encountered problems in steroid hydroxylation, but this can be controlled to a certain extent by appropriate choice of fermentation conditions, usually involving low substrate concentrations and minimization of fermentation time. Some microorganisms are... 

The principles alluded to above are exemplified by some further steroid hydroxylations in Sections 1.4.4.2.4andl.4.4.2.5. 

A bacterial mediane noonooxygenase that catalyzes the hydroxylation of saturated hydrocarbons has been isolated, but there are severe limitations in practical use. Similarly the enzymes involved in steroid hydroxylation have been isolated and used in but this practice is not sufficiently well... 

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