Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies

Davydov, Roman; Strushkevich, Natallia; Smil, D.; Yantsevich, A. V.; Гилеп, А. А.; Usanov, Sergey A.; Hoffman, Brian M.

doi:10.1021/acs.biochem.5b00903

Cited by 34 publications

(52 citation statements)

References 36 publications

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“…Our results do not rule out a ferric peroxide mechanism, nor do they define the fraction of the normal reaction that is catalyzed by each of the two mechanisms, if both are operative. If further research implicates compound I in this reaction, then few strong cases for P450 ferric peroxide chemistry will remain, at least in the field of steroid metabolism (33,64,78).…”

Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 99%

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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We propose three mechanisms that can involve the FeO 3؉ entity and that explain the 18 O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17␣-hydroxylated products to yield 16,17␣-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6␤-hydroxylation of 16␣,17␣-dihydroxyprogesterone and the oxidation of both 16␣,17␣-dihydroxyprogesterone and 16␣,17␣-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17␣,20-lyase reaction cannot be excluded. Cytochrome P450 (P450)3 enzymes catalyze oxidations of more chemicals than any other group of proteins (1). The list of reactions includes aliphatic and aromatic hydroxylations, heteroatom oxidations, epoxidations, and reactions involving both ring formation and cleavage (2-4). Many P450 reactions are important in the biosynthesis and degradation of steroids and sterols (4, 5), including several critical C-C bond cleavage reactions, i.e. those catalyzed by P450s 11A1, 17A1 (Fig. 1), 19A1, and 51A1 (6, 7).The mechanisms of the C-C cleavage reactions have been the subject of considerable interest and debate. One of the questions with P450s 17A1, 19A1, and 51A1 has been whether the active oxidant is a ferric peroxide (FeO 2 Ϫ ), which is an early intermediate following oxygen addition to the iron (Fig. 2, step 4) or the FeO 3ϩ species (Fig. 2, step 6), often referred to as compound I (4, 10, 11). With P450s 17A1 and 19A1, a variety of approaches has been applied, including theoretical calculations, biomimetic models, spectroscopy, substrate atom labeling, and kinetics (12-32).These C-C bond cleavage reactions are complex, and many of the results are ambiguous; also, a "mixed" mechanism would not be discerned in many of these experiments. One powerful approach originally used by Akhtar and co-workers (27-31) analyzes the actual reaction and can provide discrimination between the nucleophilic FeO 2 Ϫ and electrophilic FeO 3ϩ reactions (Fig. 2), based on the incorporation of 18 O label from O 2 into the carboxylic acid products (Fig. 3) (7). However, these experiments are complicated due to the ubiquitous presence of formic acid (P450 19A1 and 51A1 reactions) and acetic acid (P450 17A1) in laboratory settings. Thus, the data from such experiments are interpreted with the most confidence when the steroid substrates are labeled with 2 H or 13 C isotopes to facilitate analysis (15,33). Even then, the mass spectrometry results can be problematic, particularly if a shift of only one atomic mass unit is introduced and isotopologues derived from 18 O incorporation are not discriminated from molecules containing natural abundance 13 C atoms (33). The incorporation of one atom of 18 O label from O 2 into formic acid (F...

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Section: B Ring Hydroxylation Of 16␣17␣-dihydroxyprogesterone By P45mentioning

confidence: 99%

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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“…To the best of our knowledge, this is the first report of formation of such dihydroxylated products by any CYP450 enzyme in a two‐step oxidation of progesterone, although Streptomyces roseochromogenes NCIB 10984 was shown to hydroxylate exogenous progesterone to 16α‐hydroxyprogesterone and subsequently to 2β,16α‐dihydroxyprogesterone . Some CYP450s, such as CYP17A1, cytochrome P450scc (CYP11A1), cytochrome P450BioI (CYP107H1), and MycG, have been known to catalyze the multistep oxidation of their substrates in a sequential manner …”

Section: Resultsmentioning

confidence: 89%

Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

et al. 2018

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CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.

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“…The multistep oxidation of steroids in the presence of CYP154C8 was not previously observed for any bacterial source of CYPs, although cytochrome P450 BioI (CYP107H1) and MycG from bacterial sources have been reported to catalyze the multistep oxidation of fatty acids and mycinamycin IV, respectively . Some CYPs such as CYP11A1, CYP17A1, CYP19A1, and CYP24A1 from mammalian sources are widely known to catalyze the multistep oxidation of steroids …”

Section: Discussionmentioning

confidence: 94%

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Dangi

Park

2018

ChemBioChem

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CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H O , with optimum conversion surprisingly being achieved at ≈75 mm H O . More importantly, H O tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H O . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

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Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies

Cited by 34 publications

References 36 publications

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

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