The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

doi:10.1016/j.jsbmb.2008.11.012

The Journal of Steroid Biochemistry and Molecular Biology

Volume 113, Issues 1–2, January 2009, Pages 98-104

https://doi.org/10.1016/j.jsbmb.2008.11.012 Get rights and content

Abstract

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase (p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity (p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.

Introduction

Androstenone is a C19 16-androstene steroid produced as a phermonal hormone by leydig cells in porcine testis from C21 precursors, pregnenolone and progesterone [1]. Androstenone is transported via the blood stream to the submaxillary salivary glands [2], where it binds to a specific binding protein which concentrates the steroid in this area [3]. Androstenone is also highly lipophilic and accumulates in the adipose tissue [4] leading to the disagreeable boar taint odour and flavour of meat from uncastrated male pigs.

The primary step in the synthesis of androstenone from pregnenolone is the formation of 5,16-androstadien-3β-ol (ANβ), which is catalyzed by the andien-β synthase system in a cytochrome P450C17 (CYP17A1) dependent reaction [2], [5]. Alternatively, pregnenolone can be converted to 17α-hydroxypregnenolone (17OHP) via the 17α-hydroxylase reaction and then via the C17,20-lyase reaction to the androgen, dehydroepiandrosterone (DHEA). CYP17A1 is thus a key enzyme that regulates the production of androgens versus 16-androstene steroids.

The involvement of CYP17A1, microsomal cytochrome b5 (CYB5A, Type I), NADPH cytochrome P450 reductase (POR) and cytochrome b5 reductase (CYB5R3) in the andien-β synthase system has been shown previously [6], [7]. Ogishima et al. [8] demonstrated that the outer mitochondrial membrane cytochrome b5 (CYB5B, Type II) is also involved in steroid hormone metabolism in rats and guinea pigs. The potential role of CYB5B in ANβ synthesis in pigs has not yet been investigated.

CYB5 has many roles such as: (a) transfer of electrons from NADH to desaturase [9], (b) NADH-dependent reduction of methemoglobin to regenerate hemoglobin [10], and (c) stimulation of cytochrome P450 dependent oxygenation [8]. The exact mechanism of CYB5 is not yet completely understood; however, POR is the primary electron donor because the ferric state of P450 is a lower redox potential than CYB5 [8]. Experiments with apo-CYB5, which lacks the heme moiety, and holo-CYB5 suggest that CYB5 is not responsible for direct electron transfer but exerts a saturable, allosteric effect on the CYP17A1–POR complex [11]. The POR functions by catalyzing electron transfer from NADPH to cytochrome P450 during catalysis [12] and is also involved in electron transfer from NADPH to heme oxygenase [13] and CYB5 [14].

NADH cytochrome b5 reductase (CYB5R3) works in a similar manner as shown below:NADH + H⁺ + 2 ferricytochrome CYB5 → NAD⁺ + 2 ferrocytochrome CYB5CYB5R3 is a flavoprotein that catalyzes the reduction of CYB5, using FAD as a prosthetic group. Two forms of CYB5R3 are known, a membrane-bound form in somatic cells and a soluble form in erythrocytes [15]. The membrane-bound form consists of both the membrane binding domain and the catalytic domain, where as the soluble form only consists of the catalytic domain [16], [17]. The role of CYB5R3 on the 17α-hydroxylase, C17,20 lyase and andien-β synthase activity of CYP17A1 has previously been investigated; CYB5R3 is not necessary for the production of 17OHP or DHEA, but CYB5R3 significantly increases andien-β synthase activity [6].

Among the several systems developed to characterize enzymatic activity, transfection of expression constructs into intact mammalian cells provides the opportunity to study the activity of the expressed proteins in the native microsomal environment and to study various combinations of enzymes, redox partners and substrates used [18], [19]. In this report, we studied the effects of CYB5A and CYB5B on the activities of CYP17A1 by transient transfection of human embryonic kidney (HEK-293FT) cells with expression constructs for CYP17A1, POR, CYB5R3 and either CYB5A or CYB5B. With this system we have the ability to over-express the proteins of interest, and to vary the relative amounts of these proteins to produce a well defined and active system with minimal interference from endogenous proteins that would occur with hepatocytes or microsomal preparations.

Our objective was to determine how CYB5A and CYB5B modulate the three activities of porcine CYP17A1: 17α-hydroxylase, C17,20 lyase and andien-β synthase activity. We were especially interested in systems that would maintain the normal production of sex steroids (through the 17α-hydroxylase and C17,20 lyase reactions) while decreasing the production of the 16-androstene steroids (through the andien-β synthase reaction), since this might lead to methods for controlling boar taint.

Section snippets

Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B

The entire coding regions of porcine POR, CYP17A1, CYB5R3, CYB5A and CYB5B were amplified from porcine testis cDNA by PCR using platinum Pfx DNA polymerase (Invitrogen) and primers listed in Table 1. The amplified segments were then cloned into pcDNA3.1/V5-His TOPO (Invitrogen) to produce expression vectors. Expression vectors for V5-His tagged proteins were generated so that the expressed proteins could be detected by Western blotting using anti-V5-HRP antibody; vectors expressing the untagged

Cloning of porcine CYB5R3 and optimization of the expression system

The nucleotide and deduced amino acid sequence of porcine CYB5R3 compared with human CYB5R3 is shown in Fig. 1. The homology between porcine and human was 90% and 93% at the nucleotide and amino acid level respectively.

We first optimized the HEK-293 system to obtain similar levels of protein expression and activity and to determine the optimum time for protein expression following transfection. Fig. 2A displays a Western blot of cells expressing V5-tagged CYP17A1, POR, CYB5R3, CYB5A and CYB5B

Discussion

The primary purpose of this study was to investigate the effect of CYB5A versus CYB5B on the activity of porcine CYP17A1 in catalyzing the synthesis of ANβ. This is the initial step in the synthesis of 16-androstene steroids, which are a major component of boar taint in entire male pigs. A more complete understanding of this process may lead to targets for selective breeding or other methods for decreasing the production of 16-androstene steroids and thereby reduce boar taint.

It has been

Acknowledgments

This work was supported by funding from the Ontario Ministry of Agriculture, Food and Rural Affairs and the Natural Sciences and Engineering Research Council of Canada Discovery Grants Program.

References (27)

R. Claus et al.
Physiological aspects of androstenone and skatole formation in boar: a review with experimental data
Meat Sci.
(1994)
D.B. Gower
16-Unsaturated C19 steroids: a review of their chemistry, biochemistry and possible physiological role
J. Steroid Biochem.
(1972)
E.J. Squires
Involvement of cytochrome P450 in the synthesis of 5,16, androstadien-3β-ol from pregnenolone in pig testis microsomes
J. Steroid Biochem.
(1989)
W.J. Meadus et al.
Cytochrome P450c17 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3β-ol from pregnenolone in the presence of cytochrome b5
J. Steroid Biochem. Mol. Biol.
(1993)
S. Nakajin et al.
Cytochrome b5 promotes the synthesis Δ16-C19 steroids by homogenous cytochrome P-450 C21 side-chain clevage from pig testis
Biochem. Biophys. Res. Commun.
(1985)
T. Ogishima et al.
Identification of outer mitochondrial membrane cytochrome b5 as a modulator for androgen synthesis in leydig cells
J. Biol. Chem.
(2003)
T. Yoshida et al.
Features of the reaction of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system
J. Biol. Chem.
(1978)
Z. Ilan et al.
Evidence for a new physiological role of hepatic NADPH: ferricytochrome (P-450) oxidoreductase
J. Biol. Chem.
(1981)
J. Ozols et al.
Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase
J. Biol. Chem.
(1985)
S. Nakajin et al.
Microsomal cytochrome P-450 from neonatal pig testis
J. Biol. Chem.
(1981)

P. Soucy et al.

Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human P450c17

J. Steroid Biochem. Mol. Biol.

(2002)

R.J. Auchus et al.

Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer

J. Biol. Chem.

(1998)

J. Babol et al.

Investigation of factors responsible for the development of boar taint

Food Res. Int.

(1996)

Cited by (24)

Structural basis of bidirectional allostery across the heme in a cytochrome P450 enzyme
2023, Journal of Biological Chemistry
Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (¹⁹F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C), and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein–protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, ¹⁹F-NMR spectra of the proximal surface indicate that ligand-induced allostery modulates the environment at the C-helix but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs.
Effect of mutations in porcine CYB5A and CYP17A1 on the metabolism of pregnenolone
2019, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
Using a comparative endocrinology approach, we identified residues in the steroid binding pocket of CYP17A1 and residues on CYB5A and CYP17A1 that may be involved in binding between these two proteins. We then expressed these mutations in HEK 293-FT cells along with porcine POR and CYB5R3 [16] and measured the formation of metabolites from radiolabelled pregnenolone by HPLC. This allowed us to identify amino acid changes that differentially affect the formation of the 16-androstene steroids and the 17α-hydroxylase and C17,20 lyase reactions that occur sequentially for the synthesis of sex steroids (DHEA).
Cytochrome P450 17A1 (CYP17A1) catalyses the 17α-hydroxylation and 17,20 lyase reactions to convert pregnenolone to 17α-hydroxypregnenolone (17OHP) and subsequently the androgen dehydroepiandrosterone (DHEA). In pigs and humans, CYP17A1 also catalyses the delta-16-synthase reaction to produce the 16-androstene steroid 5,16-androstadien-3β-ol (16A), which is then further metabolised to the sex pheromone androstenone. Cytochrome b5A (CYB5A) stimulates the 17,20 lyase reaction and is required for the delta 16-synthase reaction. We have identified and mutated residues in porcine CYP17A1 and CYB5A that may alter the synthesis of DHEA and 16A. This included residues in the steroid binding pocket of CYP17A1 and residues on the surface of CYP17A1 and CYB5A that are involved in binding of CYP17A1 to CYB5A. We then expressed the various mutations of CYB5A and CYP17A1 along with porcine cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase (CYB5R3) in HEK293 cells and measured the formation of metabolites 16A, 17OHP and DHEA from radiolabelled pregnenolone by high performance liquid chromatography (HPLC). Mutations were identified in both CYP17A1 and CYB5A that affected the production of the different metabolites and also affected the overall production of metabolites. Several combinations of mutations decreased the production of both 16A and DHEA and increased production of 17OHP, while the N62S mutation of CYB5A with wild type CYP17A1 increased production of both 16A and DHEA. The best combination of mutations to reduce the production of 16A, while maintaining the production of DHEA and the overall conversion similar to wild type are the N21K, L28V, N21K/L28V and the R52 M/N62S mutations of CYB5A with the D103S mutation of CYP17A1.
The sulfoconjugation of androstenone and dehydroepiandrosterone by human and porcine sulfotransferase enzymes
2018, Steroids
Citation Excerpt :
Amplified DNA fragments containing appropriate start codon and Kozak sequence (GCC) were gel-purified using a PureLink quick gel extraction kit (Invitrogen) and were subcloned into mammalian expression vector pcDNA3.1/V5-His-TOPO using a TA cloning kit (Invitrogen). Expression vectors for V5-His tagged proteins were generated in order to detect protein expression by Western blot using anti-V5-horseradish peroxidase (HRP) antibody [16]. Sequences of all constructs were confirmed by DNA sequence analysis.
Porcine sulfotransferase 2A1 (pSULT2A1) is a key enzyme involved in the testicular and hepatic sulfoconjugation of steroids such as dehydroepiandrosterone (DHEA) and potentially androstenone. This latter steroid is a major cause of boar taint, which is an unpleasant off-odour and off-flavour in pork from male pigs. Sulfotransferase 2B1 (pSULT2B1) may also be important, although no direct evidence exists for its involvement in sulfoconjugation of steroids. The purpose of this study was to investigate the sulfoconjugation activity of human and porcine sulfotransferases towards DHEA and androstenone. pcDNA 3.1 vectors expressing porcine (p) SULT2A1, pSULT2B1, human (h) SULT2A1, hSULT2B1a, and hSULT2B1b enzymes were transfected into human embryonic kidney cells. Transfected cells were then incubated with either androstenone or dehydroepiandrosterone (DHEA) in both time-course and enzyme kinetics studies. The production of sulfonates of androstenone metabolites and DHEA sulfonate increased over time for all enzymes with the exception of pSULT2B1. Enzyme kinetics analysis showed that androstenone and DHEA were poor substrates for the human orthologs, hSULT2B1a and hSULT2B1b. Human and porcine SULT2A1 showed substantially different substrate affinities for androstenone (K_m 5.8 ± 0.6 µM and 74.1 ± 15.9 µM, respectively) and DHEA (K_m 9.4 ± 2.5 µM and 3.3 ± 1.9 µM, respectively). However, these enzymes did show relatively similar sulfonation efficiencies for DHEA (V_max/K_m 50.5 and 72.9 for hSULT2A1 and pSULT2A1, respectively). These results highlight the species differences in sulfonation activity and provide direct evidence, for the first time, suggesting that pSULT2B1 is not involved in sulfonation of either androstenone metabolites or DHEA.
Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis
2018, Journal of Steroid Biochemistry and Molecular Biology
Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.
The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1)
2015, Journal of Steroid Biochemistry and Molecular Biology
The steroid hydroxylation and carbon–carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F–G loop and the B’-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized.
This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.
Pathways and genes involved in steroid hormone metabolism in male pigs: A review and update
2014, Journal of Steroid Biochemistry and Molecular Biology
This paper reviews state-of-the-art knowledge on steroid biosynthesis pathways in the pig and provides an updated characterization of the porcine genes involved in these pathways with particular focus on androgens, estrogens, and 16-androstenes. At least 21 different enzymes appear to be involved in these pathways in porcine tissues together with at least five cofactors. Until now, data on several porcine genes were scarce or confusing. We characterized the complete genomic and transcript sequences of the single porcine CYP11B gene. We analyzed the porcine AKR1 gene cluster and identified four AKR1C, one AKR1C like genes and one AKR1E2 gene. We provide evidence that porcine AKR1C genes are not orthologous to human AKR1C. A new nomenclature is thus needed for this gene family in the pig. Thirty-two genes are now described: transcript (30 + 2 characterized in this study) and genomic (complete: 18 + 1 and partial: 12 + 1) sequences are identified. However, despite increasing knowledge on steroid metabolism in the pig, there is still no explanation of why porcine testes can produce androstenone and epiandrosterone, but not dihydrotestosterone (DHT), which is also a reduced steroid.

View all citing articles on Scopus

View full text

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Abstract

Introduction

Section snippets

Construction of expression vectors for POR, CYP17A1, CYB5R3, CYB5A and CYB5B

Cloning of porcine CYB5R3 and optimization of the expression system

Discussion

Acknowledgments

Meat Sci.

J. Steroid Biochem.

J. Steroid Biochem.

J. Steroid Biochem. Mol. Biol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

Investigation of factors responsible for the development of boar taint

Food Res. Int.