Transformation Of Steroids Research Articles

Tracing the past half century of my microbial transformation studies

In the middle of 1950's, microbial transformation technology was introduced into the field of synthetic chemistry as a new methodology. There was a sudden interest in research on the problems of producing steroid hormones by microbial transformation. At that time, the first project entitled "The Study for Microbial Transformation of Steroids", the "Tsuda Project", was established in the Institute of Applied Microbiology (IAM), University of Tokyo, in the spring 1956, in which I took part. This paper summarizes a number of results of our microbial transformation reactions not only in the synthesis of steroidal compounds, but also more broadly for other organic compounds, such as pravastatin, etc. The results are divided into five categories: 1) Microbial transformation of steroids, 2) Correlation between isolation sources of Pseudomonas spp. and their transformation activities, 3) Fermentation Production of prednisolone by Bacillus pulvifaciens SANK 71760, 4) Microbial transformation of siccanin, and 5) Development and fermentation production of pravastatin. About 30 years later, almost at the end of my microbial transformation studies, I had the opportunity to find some microbial strains having superior hydroxylation ability of ML-236BNa to pravastatin. Fortunately, Streptomyces carbophilus SANK 62585 was finally selected as a potent microbial converter with the formation of a lesser amount of by-products. With the view of industrial production of pravastatin, many studies and improvements were made to the culturing conditions to obtain productivity available commercially.

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C 6, C 7, C 11 and C 14, resulting in formation of 6 β-, 7 α-, 11 α- and 14 α-hydroxy-AD. One strain also produced small amounts of 6 β,14 α-dihydroxy-AD. Partly, the 6 β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ 4-double bond was observed, leading to the formation of 5 α-androstane derivatives. In minor yields the carbonyl functions at C 3 and C 17 were reduced leading to the formation of 3 ξ-OH or 17 β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.

Cortexolone 11 β-hydroxylation in protoplasts of Curvularia lunata

Transformation of cortexolone to hydrocortisone by the filamentous fungus Curvularia lunata using protoplasts as a research tool was studied. A stable mutant of C. lunata (IM 2901/366) hydroxylated cortexolone at 11 β-position with significantly higher activity than the wild strain (IM 2901) at the similar rate of growth. Protoplasts released from the mycelium of the wild strain and the mutant transformed the steroid substrate with about four and over five times higher yield than the parental mycelium (respectively). Due to the higher hydroxylation ability of the mutant, the activity of mutant protoplasts was 24 times higher than the activity of the mycelium of the wild strain. Hydroxylation of cortexolone was blocked in both strains by the inhibitor of cytochrome P-450 (cyt P-450) activity, ketoconazole. Cyt P-450, as hemoprotein involved in steroid-hydroxylating system, was localized in microsomal fractions of both strains. Estimation of cyt P-450 content in the intact protoplasts revealed that the level of cyt P-450 in the mutant cells was higher and it increased more rapidly in steroid presence than in the wild strain cells. It is suggested that the higher amount of cyt P-450 and higher steroid transformation activity of the mutant result from mutation(s) of genes involved in steroid 11 β-hydroxylase synthesis regulation.

Microbial hydroxylation of 13 β-ethyl-4-gonene-3,17-dione

Among the microbiological transformations of steroids 15 α-hydroxylation of 13 β-ethyl-4-gonene-3,17-dione is an industrially important one [Von H. Hofmeister, K. Annen, H. Laurent, K. Petzoldt, R. Wiechert, Arzneim.-Forsch. 36 (1986) 781], since it results in an intermediate of the synthesis of Gestoden, a widely used contraceptive drug. The aim of our research was to select fungal strains for hydroxylation of 13 β-ethyl-4-gonene-3,17-dione which produce the 15 α-hydroxylated product in a high yield. According to our taxonomical studies, several species of Aspergillus, Fusarium, Mortierella, and Penicillium genera fulfill this requirement. It has been reported that the 15 α-hydroxylating enzyme of Penicillium raistrickii is inducible by various steroidal compounds [S. Irrgang, D. Schlosser, H.-P. Schmauder, Biotechnol. Lett. 14 (1992) 33]. We found that the enzyme of Fusarium nivale (VJ-63 strain) is also advantageously induced by norethisterone, which significantly increased the economic efficiency of this biotransformation process [A. Jekkel, É. Ilkőy, J. Sütő, G. Ambrus, Gy. Horváth, I. Bősinger, I. Pallagi, I. Láng, E. Gyepessy, Hungarian Patent Appl. P-9602249 (1996)].

DHEA and the Intracrine Formation of Androgens and Estrogens in Peripheral Target Tissues: Its Role during Aging

Human and some other primates are unique since their adrenals secrete large amounts of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), which are converted into androstenedione (4-dione) and then into potent androgens and estrogens in peripheral tissues, therefore providing autonomous intracrine control to target tissues that can adjust the formation and metabolism of active sex steroids according to local requirements. Knowledge in this area has recently made rapid progress with the elucidation of the structure of most of the tissue-specific cDNAs and genes that encode the steroidogenic enzymes responsible for the transformation of these inactive precursor steroids into androgens and/or estrogens. It is estimated that 30 to 50% of total androgens in men are synthesized in peripheral intracrine tissues from inactive adrenal precursors while, in women, peripheral estrogen formation is even more important, the best estimate being 75% before menopause and 100% after menopause. The marked reduction in the formation of DHEA-S by the adrenals during aging, especially before the age of 50 years, results in a dramatic fall in the formation of active sex steroids in peripheral target tissues, a situation which is thought to be associated with a long series of age-related decreases such as insulin resistance, obesity, osteoporosis, cardiovascular diseases, loss of muscle mass, cancer and other diseases. We have demonstrated for the first time a series of medically important beneficial effects of DHEA administered for 12 months to post-menopausal women. Most interestingly, the bone mineral density significantly increased. This relatively rapid change was associated with an increase in plasma osteocalcin, a marker of bone formation, while a decrease in bone resorption reflected by a decrease in urinary hydroxyproline excretion was observed in parallel. In addition, the estrogenic stimulation of vaginal cytology in the absence of any sign of stimulatory effect on the endometrium is also of potentially major interest for the prevention and management of menopause. Furthermore, the inhibitory effect of DHEA on the growth of human breast cancer xenografts in vivo in nude mice supports the beneficial use of DHEA as hormone replacement therapy in women.

An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17alpha-hydroxylase-17,20-lyase (P-45017alpha also CYP17).

Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone-->17alpha-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17alpha-hydroxypregnenolone-->dehydroepiandrosterone and 3beta-hydroxyandrost-5-ene-17beta-carbaldehyde-->3beta-hydroxya ndrost -5,16-diene+androst-5-ene-3beta,17alpha-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the t12 of the P-450-->P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for 'activating' water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

Microbial 9α-hydroxylase: Epoxidation of 9(11)-dehydro-17α-methyl-testosterone

Steroid 9alpha-hydroxylase is a key enzyme system in steroid nucleus degradation in company with Delta(1)-dehydrogenase. To examine 9alpha-hydroxylase activity during microbial transformation of steroids, 9(11)-dehydro-17alpha-methyl-testosterone was adopted as a stable substrate for preventing the rupture of steroid nucleus. UsingNocardia restrictus ATCC 14887 capable of introducing a 9alpha-hydroxyl group into steroids, 9alpha, 11alpha-oxido-17beta-hydroxy-17alpha-methyl-4-androstene-3-one and 9alpha, 11alpha-oxido-17beta-hydroxy-17alpha-methyl-1, 4-androstadiene-3-one were obtained. These microbiologically transformed products could be used as reference compounds in the enzyme assay.

Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii.

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

Bacterial cytochromes P-450.

The cytochromes P-450 (P-450s) constitute an extremely large family ('superfamily') of haemoproteins that catalyse the oxidation of a wide range of physiological and non-physiological compounds. A remarkable feature of the P-450s is the manipulation of the same basic structure and chemistry to achieve an enormous range of functions in organisms as diverse as bacteria and man. Indeed, the P-450s have been described as 'the most versatile biological catalyst known'. Much research is focussed on mammalian P-450s, with their roles in such processes as steroid transformations and the metabolism of carcinogens and other xenobiotics. However, our knowledge of the structure and function of the P-450s has been advanced by analysis of a limited number of its bacterial members, primarily P-450cam from Pseudomonas putida. Four P-450 structures have been solved to date, all of which are from bacterial sources. The aim of this review is to assess current knowledge of the many bacterial P-450s, with emphasis on their diverse biological roles and on the advances in our knowledge of this extremely important enzyme class, which have been made feasible through their study.

Microbial transformations of steroids--X. Cytochromes P-450 11 alpha-hydroxylase and C17-C20 lyase and a 1-ene dehydrogenase transform steroids in Nectria haematococca.

Nectria haematococca contains four enzymes that metabolise exogenous steroids. Two of these are microsomal cytochromes P-450 which act sequentially on progesterone producing firstly, by side-chain cleavage, the C19 steroid androstenedione (C17-C20 lyase), and then, in a subsequent set of transformations, 11 alpha-hydroxylated derivatives (11 alpha-hydroxylase). Two other conversions occur after side-chain cleavage. Unsaturation, in the form of a double bond at C1-C2, is introduced into the A ring by a catalytically self-sufficient microsomal 1-ene dehydrogenase. This enzyme is specific for C19 substrates. A C17-specific oxidoreductase is also involved in the production of androstenedione and testosterone from progesterone. The lyase, 11 alpha-hydroxylase and 1-ene dehydrogenase were purified to homogeneity.

Effect of two levels of transgalactosylated oligosaccharide intake in rats associated with human faecal microflora on bacterial glycolytic activity, end-products of fermentation and bacterial steroid transformation.

The effects of two levels of transgalactosylated oligosaccharide (TOS) intake on bacterial glycolytic activity, end products of fermentation and bacterial steroid transformation were studied in rats associated with a human faecal flora. Rats were fed a human-type diet containing 0, 5 or 10% TOS. Caecal pH decrease correlated with the amount of TOS in the diet. Intake of the TOS diet induced a decrease in blood cholesterol and a strong increase in beta-galactosidase activity in the hindgut. TOS fermentation led to production of hydrogen and short chain fatty acids, whereas ammonia and branched-chain fatty acids were decreased. A diet containing 10% TOS increased caecal lactic acid concentrations and reduced beta-glucuronidase activities and steroid transformation.

Steroid transformations in pregnant mares: Metabolism of exogenous progestins and unusual metabolic activity in vivo and in vitro

The mare possesses unique steroid hormone metabolic activity during pregnancy in that peripheral 4-pregnene-3,20-dione (progesterone; P4) is undetectable by 220 days gestation. This study examines in vivo metabolism of progestins by the pregnant mare and in vitro metabolic activity of maternal and fetal tissues. Pregnant mares (n = 3) received intravenous infusions of 3β-hydroxy-5-pregnen-20-one (pregnenolone; P5), P4, 5α-pregnane-3,20-dione (5α-DHP), 3β-hydroxy-5α-pregnan-20-one (3β-5α), deuterium labeled (D4)-P5, D4-3β-5α and vehicle. Anestrous mares (n = 2) were infused with P5, P4, and vehicle. Also, placenta, endometrium, fetal gonad, maternal and fetal liver, and adrenal from 4 animals were incubated in D4-P5, D4-5α-DHP, or D4-3β-5α. Pregnant mares (in vivo) converted infused P5 predominantly to 5-pregnene-3β,20β-diol (P5-ββ), 5α-DHP, 20α-hydroxy-5α-pregnan-3-one (20α-5α), and 3β-5α while only minor concentrations of P4 were detected. Infused P4 was converted primarily to 5α-DHP and 20α-hydroxypregnanes and when 5α-DHP served as a substrate, other 5α-pregnanes were formed. Infused 3β-5α was either reduced to βα-diol or oxidized to 5α-DHP. Regardless of treatment, anestrous mares were incapable of producing any 20α-hydroxypregnanes but could convert P5 to P5-ββ and P4 in quantities similar to that of pregnant mares. In vitro, placenta converted D4-P5 to D4-P4 while D4-3β-5α was oxidized to D4-5α-DHP. Endometrium converted substrate primarily to D4-20α-hydroxypregnanes. Both maternal and fetal liver produced D4-20β-hydroxy compounds regardless of substrate. Maternal and fetal adrenal were capable of conversion of D4-P5 to D4-P4 while fetal gonad did not perform any significant metabolism of substrate, though it produced P5. These data explain the absence of P4 and presence of other progestin metabolites in maternal circulation during mid- and late pregnancy. Pregnenolone can be 5α-reduced to 3β-5α, and 3β-5α could be 3-oxidized to 5α-DHP. It is 5α-DHP that may serve as substrate for other 5α-pregnanes.

Novel microbial transformations of steroids.

In recent years, reactions catalyzed by microbes or their enzymes have been extensively evaluated from the viewpoint of synthetic organic chemistry. These biocatalysts offer greater specificity than conventional organic reactions and hence used as reagents to carry out certain specific chemical reactions. Microbes find use in the synthesis and production of pharmacologically active steroids. Microbial transformation of steroids was recognised after the successful llα-hydroxylation of progesterone by the fungus, Rhizopus arrhizus.1 Microorganisms can be efficiently used to carry out hydroxylation of a steroid molecule at specific positions. A significant amount of work has already been carried out on the microbial steroid transformations.2–4. We isolated two versatile microorganisms identified as Mucor piriformis and Moraxella sp. that carry out novel and preparatively useful transformations of some steroids. The unique properties of these two microorganisms as biocatalyst are presented.

Steroid transformation by mutants of Mycobacterium sp. with altered response to antibiotics.

Mycobacterium sp. NRRLB3683 which is capable to convert beta-sitosterol to 1,4-androstadiene-3,17-dione (ADD) was treated with methyl methane sulfonate and two strains with altered sensitivity to various antibiotics were obtained. One of the strain was steroid 1(2)-dehydrogenase negative and the other positive. Efficiency of utilization of sterols followed the order beta-sitosterol > cholesterol > soluble cholesterol. The steroid 1(2)-dehydrogenase negative strain was capable of producing 17KS (AD) from beta-sitosterol and converting AD to testosterone and ADD to AD suggesting the negative role of 1(2)-dehydrogenase in sterol side chain cleavage and decrease in hydrogenase activity by mutation. But this enzyme can perform the reverse reaction under aerobic condition.

Microbial transformation of steroids: Contribution to 14α-hydroxylations

The regioselective and stereoselective hydroxylation of steroids by fungal strains previously known for their hydroxylation capabilities, such as Thamnostylum (= Helicostylum) piriforme ATCC 8992, Mucor griseocyanus ATCC 1207a, Actinomucor elegans (= Mucor parasiticus) MMP 3122 ( Mucorales), and Zygodesmus sp. ATCC 14716, was investigated with special interest for the 14α-hydroxylation reaction. A preliminary screening had shown that some of these microorganisms were adequate for the production of 14α-hydroxylated derivatives of the following steroids: progesterone, 5β-pregnane-3,20-dione, 3β-hydroxy-5β-pregnane-20-one, 3β-hydroxy-5β-17(αH)-etianic acid methyl ester, androst-4-ene-3,17-dione, and testosterone. About 20 metabolites have been isolated and purified by silicagel chromatography and semi-preparative reverse-phase HPLC. These metabolites have been fully characterized by 1H, 13C NMR and mass spectrometry. All the identified metabolites were hydroxylated at some distinct positions, such as 6β-, 7α-, 9α-, 14α-, 15β-, or dihydroxylated at 6β,14α-, 7α,14α-, 9α,14α-, 14α,15α-, 14α,15β-positions; nine of these metabolites have not been reported previously. The relationship between the structural features of the investigated steroids and the site-specific hydroxylation has been delineated, and progesterone was found to be the best substrate for the production of 14α-hydroxylated derivative, using T. piriforme.

Microbial transformation of steroids—IX. Purification of progesterone hydroxylase cytochrome P-450 from Phycomyces blakesleeanus

Progesterone hydroxylase cytochrome P-450 was purified to homogeneity from Phycomyces blakesleeanus microsomes by a four step procedure. An M(r) value of 60,000 was determined for this protein by SDS-PAGE. The DEAE-cellulose and Blue-1 MIMETIC affinity fractions gave major peaks at 452 nm in a dithionite-reduced, carbon monoxide, difference spectrum. NaIO4-dependent progesterone hydroxylation was obtained by the pure enzyme without NADPH and NADPH-cytochrome P-450 reductase. NADPH-dependent hydroxylation required the addition of other Phycomyces microsomal proteins present in the Blue-1 fraction.

Microbial transformations of steroids—VIII. Transformation of progesterone by whole cells and microsomes of Aspergillus fumigatus

The filamentous fungus, Aspergillus fumigatus, efficienyly hydroxylated exogenous progesterone producing, after 3 h of incubation, 11α- and 15β-hydroxyprogesterone as major products. 7β-hydroxyprogesterone as a minor product and trace amounts of 7β,15β- and 11α,15β-dihydroxyprogesterone. After 72 h the dihydroxyprogesterones were the sole metabolites in the culture medium. Microsomes, prepared by Ca 2+ precipitation, catalysed only monohydroxylation of progesterone at the same sites as whole cells. Hydroxylation was dependent on NADPH (but not NADH) which was replaceable by NaIO 4. Hydroxylation was inhibited by carbon monoxide and by the azole fungicide, ketoconazole. Microsomes gave a dithionite-reduced, carbon monoxide difference absorbance spectrum with a peak at 448 nm and a Type-I progesterone-binding spectrum typical of cytochrome P450 interaction with substrate. Ketoconazole inhibition studies suggest the presence of two non-inducible cytochrome P450 progesterone hydroxylases, one possessing 7β site-selectivity, the other 11α/15β site-selectivity.

Microorganisms as reagents for transformations of 5?-steroids

Microbial activation of rings A, B, and C of 5α-steroids is reviewed in connection with the prospects for the use of 5α-steroids as a new starting material for the manufacturing of steroidal medicinal preparations. The combination of chemical synthesis and microbial methods of steroid transformation allows the successful solution of the problem of selective functionalization of steroid molecules, provided the suitable microorganisms are chosen and the conditions of their cultivation are optimized.

A transition from ionic to free-radical mechanisms in chemistry and enzymology

Abstract

Steroid metabolism with intestinal microorganisms

As a result of the metabolic activities of numerous anaerobic microorganisms with sterols, bile acids and steroid hormones as substrates in connection with the enterohepatic circulation of these compounds, the intestine may be considered as an "endocrine" active site or organ. The review summarizes transformations of steroids by anaerobic intestinal bacteria, the physiological and supposed pathophysiological meaning thereof. The aim is to recommend further investigation in this field with respect to both the elucidation of the reactions and biological responses.