Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1. O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

Yoshimoto, F. K.; González, E.; Auchus, Richard J.; Guengerich, F. Peter

doi:10.1074/jbc.a116.732966

Cited by 26 publications

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“…P450 17A1 17␣,20-lyase activity (13)(14)(15)). We examined P450 21A2 for a possible effect, under a variety of conditions of cytochrome b 5 concentration.…”

Section: Lack Of Stimulation By Cytochrome Bmentioning

confidence: 99%

“…The significance of this attenuation is not clear, but cytochrome b 5 was clearly not stimulatory and does not appear to be a factor influencing in vivo activity, in contrast to P450 17A1 (16,17). In the way of a caveat, we only analyzed the effect of cytochrome b 5 under the typical reconstitution conditions used with P450 21A2 and in other systems (13)(14)(15) and cannot exclude the possibility that another system might show different effects.…”

Section: Lack Of Stimulation By Cytochrome Bmentioning

confidence: 99%

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Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Wang¹,

Pallan

Zhang

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeCytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17␣-hydroxyprogesterone (17␣-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17␣-OH-progesterone). We found that the 17␣-OH-progesterone-and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either saltwasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (K s ) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b 5 , which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (> 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d 3 -progesterone, indicating that C-H bond breaking is a ratelimiting step over a 10 4 -fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting-and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.Cytochrome P450 (P450 or CYP) 5 21A2 (1), an enzyme located in the adrenal cortex, catalyzes the 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone (17␣-OHprogesterone), forming 11-deoxycorticosterone and 11-deoxycortisol, respectively (2-4). Deficiencies in the CYP21A2 gene are common and are involved in ϳ95% of the cases of congenital adrenal hyperplasia (CAH) (2, 3, 5-7), one of the most common inborn errors of metabolism. The incidence of CAH is ϳ1:15,000 worldwide, with well over 100 different genetic variants having been diagnosed. The phenotypes are generally classified as nonclassical (NC), simple virilizing (SV), and salt-wasting (SW), in order of increasing severity (2, 3, 6). The great majority of mutations in CYP21A2 can be traced to the nonfunctional pseudogene of P450 21A2 (CYP21A1P), which is found in Ͼ 70% of all Caucasians. The pseudogene is 98% identical to the functional gene, and crossovers, sequence exchanges, and apparent gene conversion between functional gene and pseudogene contribute to frequent variations in the P450 21A2 protein and the occurrence of CAH (2, 3, 6, 7).We published a crystal structure of bovine P450 21A2 bound with the substrate 17␣-OH-progesterone (8), which prov...

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“…P450 17A1 17␣,20-lyase activity (13)(14)(15)). We examined P450 21A2 for a possible effect, under a variety of conditions of cytochrome b 5 concentration.…”

Section: Lack Of Stimulation By Cytochrome Bmentioning

confidence: 99%

Section: Lack Of Stimulation By Cytochrome Bmentioning

confidence: 99%

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Wang¹,

Pallan

Zhang

et al. 2017

Journal of Biological Chemistry

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“…Here, CYP154C8 might have been involved in the degradation of steroids with hydroxyacetyl and hydroxyl functionalities at C17 by cleaving the C-C bonds at different positions. The products dehydroepiandrostenedione and androstenedione formed by lyase activity of CYP17A1 were also further hydroxylated at 16a-position [24,[42][43][44]. Steroids are ubiquitous growth substrates for microorganisms, and bacterial hydroxylase plays an important role in the degradation of steroids [38].…”

Section: Discussionmentioning

confidence: 99%

“…This challenging reaction has currently gained considerable interest due to the broad arrays of applications, which include the production of anticorrosion agents, pharmaceutical compounds, pesticides, important chemicals with biofuel applications, bioremediation, and biodegradation. More importantly, despite the prevalence of lyase activity in the biological system, the mechanism behind it has not been understood clearly [22][23][24][25].…”

mentioning

confidence: 99%

Bacterial CYP154C8 catalyzes carbon‐carbon bond cleavage in steroids

Dangi

2018

FEBS Letters

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Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C–C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR‐based structural elucidation of reaction products reveals 21‐hydroxyprednisone as the major product for prednisone, while the other product is identified as 1‐dehydroadrenosterone obtained due to C–C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C–C bond cleavage products.

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“…19 Sites of oxidation are indicted in red. The site of oxidation in the structure in the lower right corner has not been ascertained.…”

Section: Figurementioning

confidence: 99%

Intersection of the Roles of Cytochrome P450 Enzymes with Xenobiotic and Endogenous Substrates: Relevance to Toxicity and Drug Interactions

Guengerich

2016

Chem. Res. Toxicol.

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112

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Today much is known about cytochrome P450 (P450) enzymes and their catalytic specificity, but the range of reactions catalyzed by each still continues to surprise. Historically P450s had been considered to be involved in either the metabolism of xenobiotics or endogenous chemicals, in the former case playing a generally protective role and in the latter case a defined physiological role. However, the line of demarcation is sometimes blurred. It is difficult to be completely specific in drug design, and some P450s involved in the metabolism of steroids and vitamins can be off-targets. In a number of cases, drugs have been developed that act on some of those P450s as primary targets, e.g., steroid aromatase inhibitors. Several of the P450s involved in the metabolism of endogenous substrates are less specific than once thought and oxidize several related structures. Some of the P450s that primarily oxidize endogenous chemicals have been shown to oxidize xenobiotic chemicals, even in a bioactivation mode.

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Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1. O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

Cited by 26 publications

References 0 publications

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Bacterial CYP154C8 catalyzes carbon‐carbon bond cleavage in steroids

Intersection of the Roles of Cytochrome P450 Enzymes with Xenobiotic and Endogenous Substrates: Relevance to Toxicity and Drug Interactions

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