Molecular mechanisms of adrenal steroidogenesis and aspects of regulation and application [Reprint 2021 ed.] 9783112563281, 9783112563274

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Frontiers in Biotransformation Vol. 3

Frontiers in Biotransformation

Volume 3

Molecular Mechanisms of Adrenal Steroidogenesis and Aspects of Regulation and Application Edited

by

K l a u s R u c k p a u l a n d H o r s t Rein

Akademie-Verlag Berlin

Gesamt-I.SBN 3-05-500456-6 Vol. 1-ISBN 3-05-500457-4 Vol. 2-ISBN 3-05-500458-2 Vol. 3-ISBN 3-05-500459-0 Erschienen im Akademie-Verlag Berlin, Leipziger Straße 3—4, 0 - 1 0 8 6 Berlin © Akademie-Verlag Berlin 1990 Printed in Germany Gesamtherstellung: Maxim Gorki - Druck GmbH, 0 - 7 4 0 0 Altenburg Lektor: Christiane Grunow Gesamtgestaltung: Martina Bubner LSV 1315 Bestellnummer: 763 950 6 (9090/3)

Preface K . RUCKPAI L a n d H .

REIN

Volumes 1 and 2 of the series "Frontiers in Biotransformation" were concerned with molecular mechanisms in the biotransformation of exogenous compounds. This may originate from the consequences of biotransformation of just these compounds leading to malignant processes such as carcinogenesis and mutagenesis which attract the interest of bioscientists of many disciplines as pharmacologists, toxicologists, physicians, biochemists and others. The proper physiological function of the cytochrome P-450 dependent biotransformation, however, is the conversion of endogenous compounds. Therefore, in discussing current problems in biotransformation one has also to include this aspect of cytochrome P-450 functions. The most extended P-450 dependent biotransformation of endogenous substrates concerns the steroids. Steroid metabolism (anabolic and catabolic processes likewise) proceeds in several tissues of the animal organism (testes, ovaries, liver and adrenals). The adrenals are of special importance in the biotransformation of steroids due to a number of reasons. (i) Adrenals have a key function in steroidogenesis. Glucocorticosteroids and mineralocorticoids represent steroid hormones of vital importance. (ii) In the adrenal steroidogenesis not only the endoplasmic but also the mitochondrial P-450 dependent systems are involved. (iii) The biochemical composition of the mitochondrial P-450 dependent enzyme system differs from the endoplasmic system in having a further electron transferring protein between the electron donating flavoprotein and the electron accepting terminal oxygenase. (iv) Differing from most liver microsomal P-450 isozymes which are characterized by a more or less broad substrate specificity, the adrenal mitochondrial steroid converting P-450 system is characterized by highly stereospecific reactions. (v) The steroidogenesis from cholesterol to Cortisol (Cortison) is distinguished by a vectorial character shuttling between the mitochondrial matrix and

I

the cytosol involving several mitochondrial and endoplasmic cytochromes P-450. The molecular mechanisms of regulating the activities of the different adrenal steroidogenic systems are just under study and remarkable progress has been achieved within the last 5—10 years, including sequence analysis, hormonal regulation and mechanisms of insertion and processing. .But what may be the most remarkable aspect in this field is that due to the enormous practical role steroids play in therapy, biotransformation was used very early for conversion and production of useful compounds. After detection of the therapeutical properties of glucocorticoids in 1948/49 by 11 I;M H and KENDALL only 4 years later the microbial catalysis was used in the pharmaceutical industry for transforming multistep chemical synthesis in only one elegant biochemical reaction step. This pioneering work was one of the milestones in introducing pragmatically based biotechnology in pharmaceutical production. The contributions of this volume try to cover that pretentious scope. The editors would like to take this opportunity of thanking the authors of this volume for their excellent contributions. Although each review has been written individually the editors hope that when taken together they will provide a coherent picture of cytochromes P-450 involved in adrenal steroidogenesis, its regulation and application.

II

Preface

Contents

Chapter 1 Cytochrome P-450 Dependent Pathways of the Biosynthesis of Steroid Hormones S. A. Usanov, V. L. Chashchin, and A. A. Akhrem Chapter 2 Enzymology of Mitochondrial Side-Chain Cleavage by Cytochrome P-450scc J . D. Lambeth

1

58

Chapter 3 Mechanisms of Regulation of Steroid Hydroxylase Gene Expression M. R . Waterman and E . R . Simpson

101

Chapter 4 Structure and Function of Adrenal Mitochondrial Cytochrome P - 4 5 0 , ! , M. Okamoto and Y . Nonaka

127

Chapter 5 Adrenal Microsomal Cytochrome P-450 Dependent Reactions in Steroidogenesis and Biochemical Properties of the Enzymes Involved therein S. Takemori and S. Kominami

153

Chapter 6 Microbial Steroid Hydroxylating Enzymes in Glucocorticoid Production R . Megges, M. Muller-Frohne, D. Pfeil, and K . Ruckpaul

204

List of Authors

244

Subject Index

247

III

Chapter 1 Cytochrome P-450 Dependent Pathways of the Biosynthesis of Steroid Hormones S . A . USAXOV, V . L . C i r A S i i e i i i N , a n d A . A . AKIIRKM

1.

Introduction

2

2.

Monooxygenase pathways in corticosteroid biosynthesis

3

3. 3.1. 3.2. 3.3. 3.3.1. 3.3.2. 3.3.3.

Mitochondrial cytochrome P-450-dependcnt monooxygenases . . . Cholesterol side chain cleavage llß-Hydroxylation Electron transfer components Adrenodoxin reductase Adrenodoxin Principles of molecular organization of mitochondrial monooxygenases and mechanism of electron transfer

7 7 16 18 20 23

Microsomal steroid transforming enzymes 21-Hydroxylase 17«- Hyd roxylase/17,20-ly ase NADPH-cytochrome P-450 reductase 3/3-Hydroxysteroid dehydrogenase/A 5 -A 4 isomerase Molecular organization and mechanism of electron transfer in microsomal monooxygenases

33 34 37 40 41

5.

Perspectives of the application of steroid transforming enzymes . .

46

6.

Concluding remarks

49

7.

References

50

4. 4.1. 4.2. 4.3. 4.4. 4.5.

25

44

1

1.

Introduction

Cholesterol utilization via several key hydroxylation steps to yield physiologically active metabolites (corticosteroids, mineralocorticoids, sex steroid hormones, bile acids, hydroxyvitamins D 3 ) has an important implication in modern science and biotechnology. Among different oxidative processes which take place in steroidogenic tissues (adrenal cortex, ovary, testis, corpus luteum, placenta) those responsible for steroid specific regio- and stereo-selective hydroxylations including cholesterol side chain cleavage and oxygenation of steroid nucleus are of special interest. Cholesterol conversion to pregnenolone via oxidative side chain cleavage is of special interest, since it was shown to be common to different steroidogenic tissues and pregnenolone is the common precursor for steroid hormones. It is thought that the cholesterol side chain cleavage reaction is the rate-limiting step in the steroid hormones biosynthesis and regulation of the level and activity of this form of cytochrome P-450 (cytochrome P-450scc) provides the general and universal mechanism for regulation of steroid hormones level. The wide interest of scientists in cytochrome P-450-catalyzed reactions is connected with certain important fundamental and practical aspects: 1. elucidation of the molecular mechanisms of steroid hormones biosynthesis and understanding of the mechanisms by which optimal concentrations of physiologically active steroid hormones are maintained; 2. understanding of the molecular basis of differences in steroidogenic cytochrome P-450 gene expression to make a correlation between deficiencies of some steroidogenic enzymes and various disease states (congenital adrenal hyperplasia): use of specific oligonucleotides to predict some inherited deficiencies of steroidogenic enzymes; 3. until now cholesterol side chain cleavage reaction has no analogues in procariots; 4. use of highly specific stereo- and regio-selective hydroxylation reactions in biotechnology to prepare steroid hormones and their derivatives. These are only some points which demonstrate the reasons why investigation of cytochrome P-450-dependent monooxygenases has become such an active area of research. Different questions of cytochrome P-450-dependent steroidogenesis have been recently reviewed: mechanism of steroidogenic electron transfer (LAMBETH et al., 1982; SIIKUMATOV et al., 1985), role of ACTH in stimulation of stereoidogenesis (KIMUKA, 1981), cellular organization of steroidogenesis (HALL, 1984), biosynthetic units, hormones (LIEBERMAN et al., 1984), regulation of stereoidogenesis (WATERMAN a n d SIMPSON, 1 9 8 5 ; WATER-

MAN et al., 1986), role of cytochrome P-450 in the biosynthesis of physiologically active compounds (JEFCOATE, 1986). In this chapter we will concentrate on the structure and function of enzymes associated with cortico-

2

S . A . USANOV, e t a l .

steroid hormones biosynthesis, the mechanism of specific protein-protein interactions, molecular organization, substrate specificity and perspectives of the application of steroid transforming enzymes in biotechnology. 2.

Monooxygenase pathways in corticosteroid biosynthesis

Most reactions in steroid hormones biosynthesis are of the monooxygenase type catalyzed by various forms of cytochrome P-450 (Fig. 1). Cholesterol, as the starting material for steroid hormones biosynthesis, is transferred from blood and largely stored as cholesterol esters in the lipid droplets in adrenal cells. The first and rate-limiting step in the steroidogenesis is the conversion of cholesterol to pregnenolone. Pregnenolone must then reach the endoplasmic reticulum membranes where it undergoes conversion of 3/S-ol structure to the ,l4-3-ketone structure, catalyzed by 3/?-hydroxysteroid dehydrogenase//!5-/!4 isomerase, cytochrome P-450 independent enzyme, to form progesterone.

HC

I

SCCI, SCC 11,11 P, 17ot AND 21 REFER TO P 4 5 0 - C A T A L Y Z E D S T E P S OF CHOLESTEROL S I D E CHAIN C L E A V A G E , 11 p - H Y D R O X Y L A S E , 1 7 o i - H Y D R O X Y L A S E AND 21 - H Y D R O X Y L A S E , R E S P E C T I V E L Y

Fig. 1. The main cytochrome P-450 dependent pathways of steroid hormones biosynthesis. seel and 11/3-cholesterol side chain cleavage and li/J-hydroxylation reactions catalyzed by cytochrome P-450scc and cytochrome P-450 11|9 from mitochondria; 17a and 21 - C 1 7 a and C21 hydroxylation reactions catalyzed by cytochrome P-450 1 7 l > and cytochrome P-450 2 1 in microsomes; s c c l l — 17a-hydroxylase and 17,20-lyase reactions catalyzed by cytochrome P-450 1 7 a in microsomes.

Pathways of Adrenal Steroidogenesis

3

Pregnenolone may be hydroxylated in microsomes to form 17a-hydroxvpregnenolone. Progesterone is usually hydroxylated at 21-position to form 11-deoxycorticosterone if 21-hydroxylase is more active than 17a-hydroxylase/17,20-lyase. Progesterone may also be hydroxylated at 17a-position if 17v-hydroxylase is too active. If 17(X-hydroxylation takes place, it must precede 21-hydroxylation. Some of the 17»-hydroxyprogesterone is converted to androstenedione by 17a-hvdroxylase/17,20-lyase activity. Thereafter, 11-deoxycortisol or 11-deoxycorticosterone moves back to mitochondria which is necessary for 11/3-hydroxylation, catalyzed by 11/?hydroxvlating cytochrome P-450 (cytochrome P-450 U ( 3 ) to form Cortisol and corticosterone respectively and for 18-hydroxylation, catalyzed by the same cytochrome P - 4 5 0 n , to give aldosterone. The mechanism by which some steroid metabolic intermediates should move back and forth between mitochondria, where some of the steroidogenic enzymes (cholesterol side chain cleavage, 11/J-hydroxvlation) were found to be localized to microsomes, where other enzymes (17 26- > 25-hydroxycholcsterol (HUME et al., 1984). The configuration of the hydroxvl group at C7 shows that 7/?-hydroxycholesterol induces a greater maximal absorbance change than floes 7a-hydroxycholesterol. Derivatives in which the side chain amino group was closer to steroid than the C 22 -position, were found to be only very weak inhibitors and did not produce spectral changes when added to hemeprotein ( S H E E T S and V I C K E R Y , 1982; S H E E T S and V I C K E R Y , 1983; S H E E T S and V I C K E R Y , 1983; V I C K E R Y and K E L L I S , 1983). Derivatives in which the amino group was attached at a greater distance from the steroid ring than the C 2 2 -position, caused a progressive decrease in inhibitory tendency and a failure to produce spectral changes. The heme appear to be located sufficiently close to this position so that the side chain of cholesterol would allow a direct a t t a c k of an iron-bound oxidant to occur during hydroxylation and side chain cleavage. I t was shown t h a t the C 22 -position of cholesterol molecule should lie 2.9 A from the iron in the enzyme-substrate complex. Cytochrome P-450scc reveals a high degree of stereospecificity. The 22S-hydroxycholesterol binds to cytochrome P-450scc less efficiently than natural intermediate — 22R-hydroxycholesterol (HEYL et al., 1986). Spin echo studies with specifically deuterated cholesterol derivatives have revealed that the 22S deuterium of 22R-hydroxycholesterol is located at approximately 4 A far from the heme iron (GROII et al., 1983). Furthermore, presence of carbon monoxide interferes with the binding of analogs with cytochrome P-450scc indicating that the 2 2 R hydroxvl of steroid might be localized in a distance of 2—3 A from the heme iron of cytochrome P - 4 5 0 (HEYL et al., 1986). The nature of cytochrome P-450scc cholesterol interactions has also been studied using phospholipid-reconstituted cytochrome P-450scc (LAMBETH et al., 1980; Y A M A K U R A et al., 1981). The use of vesicle-reconstituted system showed t h a t only cholesterol which was incorporated in the same membrane as cytochrome P-450scc was rapidly metabolized to pregnenolone, while cholesterol in different vesicle membranes was not accessible to cytochrome P-450scc (SEYBERT et al., 1979). Several lines of evidence indicate t h a t the substrate binding site of cytochrome P-450scc is faced to the phospholipid membrane (SEYBERT et al., 1979). This is consistent with the finding t h a t externally added cholesterol is not utilized b y the intact mitochondria for stero-

10

S . A . USANOV e t a l .

idogenesis. Only cholesterol molecules of the inner mitochondrial membrane, but not completely, are accessible to cytochrome P-450scc (Cheng et al., 1985). An important prerequisite for a better understanding of the catalytic mechanism was provided by the elucidation of the primary structure of bovine cytochrome P-450scc by protein sequencing (Chasiiciitn et al., 1986) as well as by sequencing its cDNA (Moroiiashi et al., 1984). Later the cDNA sequence of human adrenocortical (Chung, 1986) and placental (Morohasiii et al., 1987) cytochrome P-450scc were elucidated. Figure 2 shows alignment of the primary structures of human and bovine cytochromes P-450scc. The amino acid sequence of human cytochrome P-450scc is 7 2 % homologous to the bovine sequence while the coding sequences are 82% homologous. Both proteins are synthesized as a precursor molecule having an extension peptide at the N-terminal sequence. Since the mature form of bovine adrenocortical cytochrome P-450scc contains He as N-terminal sequence (Akiirem et al., 1980) this means that the extra peptide consists of 39 amino acids. Human cytochrome P-450scc contains 521 amino acids and appears to have an insertion of one extra amino acid (His) compared to bovine cytochrome P-450scc. Alignment of the primary structure of bovine cytochrome P-450scc with sequences of some steroid-binding proteins indicates the homology in amino acid sequences which was interpreted as the cholesterol binding site of cytochrome P - 4 5 0 s c c : I T N V M F G

E R L G M

( G o t o i i e t al., 1 9 8 5 ) .

Besides the substrate binding site, cytochrome P-450scc contains some functionally important units: (i) the catalytic center with protoporphyrine I X as prosthetic group; (ii) the site responsible for interaction of cytochrome P-450scc with adrenodoxin; (iii) hydrophobic regions responsible for interaction with phospholipid membrane. Limited proteolysis proved to be a useful technique to study the correlation between the structure and function of cytochrome P-450scc. This method promotes determination of compact globular structures in protein molecules which possess a definite function. Indication of the domain-like structure of cytochrome P-450scc was derived from limited trypsinolysis studies. In the presence of trypsin cytochrome P-450scc disappears with concomitant appearance of two polypeptide fragments, F j and F 2 with 29.8 kDa and 26.6 kDa, respectively (Fig. 3). These fragments are relatively stable in further proteolytic modification. Prolonged incubation of cytochrome P-450scc with trypsin was shown to result in a third stable polypeptide fragment referred to as F 3 with 16.8 kDa with concomitant loss of F 2 fragment, indicating that F 3 is the further proteolytically modified fragment F 2 ( A k h r e m e t al., 1 9 8 0 ; A k i i r e m e t al., 1 9 8 0 ; C i i a s h c i i i n e t al., 1 9 8 4 ; C h a s h -

chin et al., 1984). The observed pattern of proteolytic modification of cytochrome P-450scc indicates that proteolytic modification results in an equimolar ratio of F j and F 2 fragments, with the total molecular weight equal to that of native enzyme, suggesting that these fragments might represent func-

Pathways of Adrenal Steroidogenesis

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