APPuID MICRoBowOY, Mar. 1972, p. 601-612Copyright i 1972 American Society for Microbiology

Vol. 23, No. 3Printed in U.S.A.

Reduction of the 20-Carbonyl Group of C-21Steroids by Spores of Fusarium solani and

Other MicroorganismsI. Side-Chain Degradation, Epoxide Cleavage,

and Substrate SpecificityROSAIRE PLOURDE, OSSAMA M. EL-TAYEB', AND HAMDALLAH HAFEZ-ZEDAN

FacuLtW de Pharmacie, Universit0 de Montreal, Montr&el, Qukbec, Canada

Received for publication 1 October 1971

The spores of Fusarium solani reduced the C20-carbonyl group, 1-dehydro-genated ring "A" and cleaved the side chain of 16a,17a-oxidopregn-4-ene-3,20-dione (16a, 17a-oxidoprogesterone)(I) to give the following products: 20a-hydroxy-16a, 17a-oxidopregn -4-en-3-one(II); 20a-hydroxy-16a, 17a-oxido-pregna-1,4-dien-3-one(III); 16a-hydroxy-17a-oxa-androsta-1,4-diene-3,17-dione (16a-hydroxy-1-dehydrotestololactone)(IV); and 16a,17ft-dihydroxy-an-drosta-1,4-dien-3-one (16a-hydroxy-1-dehydrotestosterone)(V). When II wasused as a substrate, it was metabolized into III, IV, and V at a slower rate thanI. Furthermore, 16a-hydroxy-androst-4-ene-3,17-dione (16a-hydroxyan-drostenedione)(X) was transformed into IV and V. Pregn-4-ene-3,20-dione(progesterone)(XII) was transformed into androsta-1,4-diene-3,17-dione (an-drostadienedione)(VIII) and 17a-oxa-androsta-1,4-diene-3,17-dione (1-dehy-drotestololactone)(IX), while 17a-hydroxy-pregn-4-ene-3,20-dione (17a-hy-droxyprogesterone)(VI) was converted into its 1-dehydro analogue (VII) withoutaccumulation of any 20-dihydro compounds. Substrate specificity in the 20-re-ductase system of F. solani, Cylindrocarpon radicicola, Septomyxa affinis, Ba-cillus lentus, and three strains of B. sphaericus are demonstrated. The 20-re-ductase is active only on steroids having the 16a, 17a-oxido, and A4-3-ketofunctions. Evidence of competition between side-chain degrading enzymes andthe 20-reductase for the steroid molecule and evidence of side-chain degrada-tion followed by epoxide cleavage (and not the reverse) are presented. A mech-anism for the epoxide opening by nongerminating spores of F. solani is postu-lated.

Several microorganisms are known to cleaveepoxides or to degrade the 17fl-acetyl sidechain of progesterone and related steroids, orboth, to give ring "D" lactones as final prod-ucts. Microbiological epoxide cleavage hasbeen reported by Camerino and Vercellone (4)and Camerino and Sciaky (3), Prochazka, et al.(18), Wix and Albrecht (34), El-Tayeb et al. (6),and Ttsmdrkeny et al. (29) using yeast cells,Rhizopus nigricans, Fusarium caucasicum,Cylindrocarpon radicicola, and Mycobac-terium phlei, respectively. Wix and Albrecht(34) reported the conversion of 16a, 17a-oxido-

' Present address: Faculty of Pharmacy, University ofCairo, Cairo, Egypt, U.A.R.

progesterone with F. caucasicum into andros-tadienedione, probably via epoxide opening,forming a 17a-hydroxy intermediate, followedby removal of side chain. On the other hand,El-Tayeb et al. (6), using C. radicicola, demon-strated the eventual conversion of the samesubstrate into 16a-hydroxy-l-dehydro-testos-terone and 16a-hydroxy-l-dehydrotesto-lolactone, probably via side-chain cleavage fol-lowed by epoxide opening (Fig. 1).

Alternative mechanisms for steroid side-chain cleavage by various organisms involve:(i) direct oxygenation at C 17-C20; (ii) reductionat C220; or (iii) hydroxylation at C 17 Petersonet al. (17) suggested that C. radicicola degrades

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PLOURDE, EL-TAYEB, AND HAFEZ-ZEDAN

CH3

OH

Rt =H (ref.34)=OH (ref.6)

FIG. 1. Conversion of 16a,17a-oxidoprogesterone into 16a-hydroxy-1-dehydrotestosterone and 16a-hy-droxy-1-dehydrotestololactone. Conversion, probably via side-chain cleavage followed by epoxide opening,demonstrated by El-Tayeb et al. (6) in C. radicicola.'

progesterone side chain via mechanism i, andthe first intermediate of the sequence, 17,3-acetoxy-androst-4-en-3-one (testosterone ace-tate), was first isolated by Fonken et al. (8)after exposure of progesterone to Clado-sporium resinae. Recently, Rahim and Sih (19)separated two enzymes from C. radicicola: anoxygenase that catalyzes the conversion ofprogesterone into testosterone acetate, whichwas then hydrolyzed by an esterase to yield17f-hydroxy-androst-4-en-3-one (testosterone).Sebek et al. (22) reported the possibility of 20fl-hydroxy derivatives (mechanism ii) as inter-mediates in side-chain degradation of proges-terone and lla-hydroxyprogesterone by Peni-cillium lilacinum. On the other hand, the pos-sibility of a 17a-hydroxy intermediate (mech-anism iii) in the oxidative cleavage of theside chain of C2, steroids has precedence inhigher organisms (5).

In previous communications (10, 12, 21, 24,25, 30), it was established that nongerminatingspores of fungi and actinomycetes can effect awide range of conversions of steroid molecules.The nongerminating spores of certain microor-ganisms showed identical (31) or modified (10,11; H. H. Zedan, M.S. thesis, Univ. of Cairo,Cairo, Egypt, 1969) activities on steroids ascompared to corresponding growing cultures.Whereas the mechanisms of steroid transfor-mation by growing cells have been extensivelystudied, Singh and Rakhit (23) appear to havebeen the first to have elucidated the mecha-nism of side-chain degradation of C-21 steroidswith the nongerminating spores of Septomyxaaffinis; the mechanism was suggested for ste-roids that do not have a 16a, 17a-epoxidegroup.A comparative study of the activities of non-

germinating spores and growing cells of F. so-lkni and other microorganisms on different

steroids has been made to determine the roleof 20-reductase in microbial side-chain degra-dation and the side-chain removal of 16a, 17a-epoxysteroids.

MATERIALS AND METHODSMicroorganisms and chemicals. Stock cultures

of C. radicicola ATCC 11011, F. solani (Faculty ofPharmacy, Univ. of Cairo), S. affinis ATCC 6737,and P. lilacinum ATCC 10114 were maintained onnutrient dextrose agar (Difco) supplemented with 1%yeast extract. Bacillus lentus ATCC 13805, and thestrains of B. sphaericus ATCC 245, 7054, and 7055were maintained on nutrient agar. They were storedat 4 C and subcultured monthly.

Analytically pure 20a-hydroxy-16a, 17a-oxido-pregn-4-en-3-one; 20a-hydroxy-16a, 17a-oxido-pregna-1, 4-dien-3-one; 16a-hydroxy-1-dehydrotes-tosterone, and 16a-hydroxy-1-dehydrotestololactonewere prepared from 16a, 17a-oxidoprogesterone,while 1-dehydrotestololactone was prepared fromprogesterone by incubation with C. radicicola aspreviously described by El-Tayeb et al. (6, 7). Otherchemicals were reagent grade.

Sporulation. Spores of F. solani and C. radicicolawere produced on Sabouraud dextrose agar, and S.affinis was sporulated on a medium consisting of 1%glucose, 0.5% yeast extract, 3% malt extract, and2.5% agar (Difco). To all the sporulation media, pro-gesterone (100 ug/ml) was added as an inducer.Abundant sporulation was attained after incubationfor 5 to 7 days at 26 to 28 C. Spores were harvestedin 1% phosphate buffer (pH 6.0), and spore suspen-sions were filtered through sterile cheesecloth,washed five times by centrifugation, resuspended inthe buffer solution, and stored at 4 C.

Steroid conversion by spores. The spores, sus-pended in the phosphate buffer, were counted in ahemocytometer, and the suspension was diluted togive 1.5 x 108 spores per ml. Transformations werecarried out in 125-ml Erlenmeyer flasks, each con-taining 25 ml of the spore suspension. Sterile glucosesolution was added to each flask to give a final con-centration of 0.5 mg/ml, and a 50 mg/ml solution of

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REDUCTION OF 20-CARBONYL GROUP OF C-21 STEROIDS

the steroid substrate dissolved in N, N-dimethylfor-mamide (DMF) was added to give a concentration of0.5 mg per ml of medium. The flasks were incubatedon a reciprocating shaker (4-cm stroke, 150cycles/min) at 26 to 28 C for 48 to 72 hr (unless oth-erwise specified).

Steroid conversion by growing cells. Transfor-mation of steroids by growing fungal mycelia wascarried out in a medium consisting of 1% glucose, 2%peptone, 1% yeast extract, and 0.25% calcium car-bonate. For transformation by bacilli, nutrient brothsupplemented with 0.5% yeast extract was used.A fresh stock culture was used to inoculate 50 ml

of the sterile medium in 250-ml Erlenmeyer flasks;the inoculated flasks were incubated on a shaker for48 hr. A 5% inoculum from the resulting growth wasused to inoculate a fresh medium, which was furtherincubated for 24 hr. A fresh flask was inoculatedwith a 5% inoculum of the resulting growth andagain was incubated for 24 hr; the substrate wasthen added, and the culture medium was incubatedas for spore-mediated transformation.

Steroid determination. All melting points weredetermined on a Rinco capillary melting point appa-ratus. Infrared (IR) spectra were recorded from KBrdiscs on a Perkin Elmer model 257 double-beam IRspectrophotometer. Ultraviolet (UV) absorptionspectra were recorded in 95% ethanol on a Unicamrecording spectrophotometer (model 800). MN-SilicaGel G/UV254 (Macherey, Nagel & Co., Germany) wasused for thin-layer chromatography (TLC) analyses.

At the end of the incubation period, the wholereaction mixture was extracted with chloroform andthe extract was dried over anhydrous sodium sulfateand evaporated to dryness under vacuum.The course of steroid transformation was followed

by TLC on Silica Gel G plates developed in ben-zene-isopropanol (6:1). The chromatoplates wereviewed under a short-wave UV lamp, sprayed with50% aqueous sulfuric acid, and heated at 110 C for10 min.When particularly specified, A4-3-ketosteroids

were differentiated from Al.4-3-ketosteroids on theplates by spraying with the strong methanolic isonic-otinic acid hydrazide (INH) solution of Smith andFoell (26); /4-3-ketosteroids give a yellow colorwithin 5 to 10 min, whereas 1 to 2 hr are required forthe same color to appear in the case of A" 4-3-keto-steroids (12). Similarly, the presence of steroidsbearing a 17fl-methyl ketone side chain was revealedon TLC plates by the nitroprusside reaction ofLisboa (13) for methyl ketones.The transformation products were separated from

crude extract by preparative TLC, recrystallized,and identified by comparison with authentic sam-ples [melting point (MP), mixed MP, IR spectra, andchromatographic properties].

For quantitative estimation of steroids, sampleswere spotted on TLC plates, developed, and air-dried. The UV absorbing spots, along with blankspots of the same size, were scraped off and elutedwith 95% ethanol. The absorption of the eluates wasmeasured spectrophotometrically at 240 nm. Resultswere expressed as percentage of total steroids.

RESULTS

Transformation of 16a ,17a-oxidoproges-terone by spores of F. solani. Spores of F.solani converted 16a,17a-oxidoprogesterone(I)to 20a-hydroxy-16a, 17a-oxido-pregn-4-en-3-one(II), 20a-hydroxy-16a, 17a-oxido-pregna-1,4-dien-3-one (III), 16a-hydroxy-1-dehydrotesto-lolactone(IV), and 16a-hydroxy-1-dehydrotes-tosterone(V).

After a 36-hr incubation period of 50 mg of Iwith spores and the usual work-up of extrac-tion, separation, and recrystallization fromacetone-petroleum ether, the following prod-ucts were obtained: 10 mg of II, 10 mg of III, 6mg of IV, and 3 mg of V. The properties of theisolated compounds were identical with thoseof corresponding authentic standards (Ta-ble 1).The relative proportions of II, III, IV, and V

varied with the period of incubation. Figure 2illustrates the course of transformation of I byspores. Substrate I disappeared gradually, andthe concentration reached a plateau at 28 hr.The four metabolites (II, III, IV, and V) showeddifferent transformation rates. As II and IIIexhibited an increase in their concentrationsfollowed by gradual disappearance, compoundsIV and V accumulated. 16-Hydroxyandrosta-1,4-diene-3, 17-dione, which might be ex-pected as an intermediate between compoundsV and IV, was not detected.When compound I was used as a substrate,

it was metabolized at a slower rate than I butwith a similar conversion pattern, whereas 16a-hydroxyandrostenedione(X) was convertedinto IV and V. Figure 3 represents the conver-sion behavior of compound II and its transfor-mation products on incubation with spores.Transformation of compound I by myce-

Hum of F. solani. Compound I was quantita-tively converted with growing F. solani into II,III, IV, and V within 12 hr (Fig. 4). Althoughthe transformation patterns of I by the sporesand the mycelium were similar, the substratewas transformed at a slightly higher rate bythe mycelium. Furthermore, lower yields of the20a-hydroxy metabolites (II and III) andhigher yields of the side-chain degradationproducts (IV and V) were obtained in the my-celial fermentation (Fig. 5).Transformation of 17a-hydroxyprog-

esterone by spores of F. solani. Spores of F.solani converted 17a-hydroxyprogesterone (VI)into 17a-hydroxypregna-1, 4-diene-3, 20-dione(VII), which was accumulated in the me-dium after a lag period of 6 hr and reacheda plateau (36%) after 72 hr, as illustrated in

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TABLE 1. Physicochemical properties of the isolated steroidsa

Compound Name Melting point INH' HSO4

II 20a-Hydroxy-16a, 17a-oxidopregn-4-en-3- 245-247 C +++ Greenish blueone

III 20a-Hydroxy-16a, 17a-oxidopregna-1,4- 225-227 C + Reddien-3-one

IV 16a-Hydroxy-17a-oxa-androsta-1, 4-diene- 207-209 C + Brown3,17-dione

V 16a, 17#-Dihydroxy-androsta-1,4-dien-3- 199-202 C + Orange brownone

VII 17a-Hydroxy-pregna-1,4-diene-3,20-dione 249-250.5 C + BrownVIII Androsta-1,4-diene-3,17-dione 139-141 C + Orange redIX 17a-Oxa-androsta-1, 4-diene-3,17-dione 219-221 C + Brown

a These properties as well as the infrared spectra were identical with those of authentic standards andthere was no depression in mixed melting points. See references 6 and 7 for more comprehensive identifica-tion of these and related steroids.

b Symbols: (+++ +), yellow color within a few minutes; (+), yellow color after 1 hr or more.

100

80

11-0

a

09

3-

IA

60

40

20

0

0..

*. /

c.~~~~~ ----L"1

74'' ' ~

4 8 1 2 16 20 24 2 8 32 36 40 48 60 72

TIMEhou rs

FIG. 2. Time course of the metabolism of 16a,1 7a-oxidoprogesterone (0) by the spores of Fusarium solani.Spores, 1.5 x 108/ml; substrate, 0.5 mg/ml; glucose, 0.5 mg/ml; pH 6.0. Symbols: 0, 20a-hydroxy-16a, 17a-oxidopregn-4-en-3-one; A, 20a-hydroxy-16a,17a-oxidopregna-1,4-dien-3-one; A, 16a-hydroxy-1-dehydrotes-tololactone; 0, 16a-hydroxy-l-dehydrotestosterone.

Fig. 6.When 200 mg of VI was incubated for 48 hr

with spores, 63 mg of VII was obtained. Com-pound VII was purified by repeated crystalliza-tion from acetone; MP found was 249 to 250.5C [reported 245 to 262 C by J. N. Gardner,U.S. Patent 3,356,696, 1967 (to Schering Corp.,Bloomfield, N.J.)], IR spectra were 2.92 um(hydroxyl), 5.88 ,um (20-ketone), 6.06, 6.2, 6.28IIm (Al'4-3-ketone). The identification of VIIwas also confirmed by a combination of micro-

biological and chemical analytical methods.Through the action of P. liiacinum ATCC10114, which brings about side-chain degrada-tion of many steroids (16), the isolated com-

pound VII was transformed into compoundVIII, identical in all respects (Table 1) with an

authentic sample of androstadienedione; an-

other compound was obtained which was ten-tatively identified as 1-dehydrotestololactone(IX). This indicates the presence of a A1'4-3-keto group in the original structure of VII. The

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REDUCTION OF 20-CARBONYL GROUP OF C-21 STEROIDS 605

o.C

0.

o-.

*..o -XE-

- O-l-a~ ~ ~ ~ *.- -

4 8 12 16 20 24 28 32 36 40 48 60 72

TIME,hou rs

FIG. 3. Time course of the metabolism of 20a-hydroxy-16a, 17a-oxidopregn-4-en-3-one (0) by the sporesof Fusarium solani. Spores, 1.5 x 108/ml; substrate, 0.5 mg/ml; glucose, 0.5 mg/ml; pH 6.0. Symbols: A, 20a-hydroxy-16a,17a-oxidopregna-1,4-dien-3-one; A, 16a-hydroxy-1-dehydrotestololactone; 0, 16a-hydroxy-1-dehydrotestosterone.

4 8 12 16 20 24

TIME ,hou rs

FIG. 4. Time course of the metabolism of 16a, 17a-oxidoprogesterone (0) by the mycelium of Fusariumsolani. The conditions are explained in the text. Symbols: 0, 20a-hydroxy-16a, 17a-oxidopregn-4-en-3-one;A, 20a-hydroxy-16a,17a-oxidopregna-1,4-dien-3-one; A, 16a-hydroxy-1-dehydrotestololactone; 0, 16a-hy-droxy-1-dehydrotestosterone.

1000

801-

60o_O..I-0

a

amI-0

401

2 01

0

1

11-0a

I..4h

80

60

20

0I I

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.o VAr

- ---& I V-- - - -0----,

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606 PLOURDE, EL-TAYEB, AND HAFEZ-ZEDAN APPL. MICROBIOL.

100

11.11180_

%.O~~~~~~~~~~~~~~~~~~~V__v-~ ....v-v

60

0-,_-- ------- IV-V0

4 8 121,6 20 24

TIME,hou rs

FIG. 5. Formation of 20a-hydroxy metabolites (20a-hydroxy-16a, 17a-oxidopregn-4-en-3-one and its 1-

dehydro analogue) and side-chain degradation products (16a-hydroxy-1-dehydrotestololactone and 16a-hy-droxy-1 -dehydrotestosterone) from l6a, 17a-oxidoprogesterone on incubation with the spores and the myce-hium of Fusarium solani. The conditions are explained in Fig. 2 and 4. Symbols: 20a-hydroxymetabolites (A)and side-chain degradation (0) products by spores; 20a-hydroxy metabolites (A) and side-chain degradation(0) products by mycelium.

100

800

60

0

I I

20

*~~~~ o----

0 j

4 8 12 16 20 24 28 32 36 40 48 60 72

TIM EhoursFIG. 6. Time course of the metabolism of 17a-hydroxyprogesterone (0) into 17a-hydroxy-pregna-1 , 4-

diene-3,20-dione (0) by the spores of Fusariumsokani. Spores, 1.5 x 108/mi; substrate, 0.5 mg/mI; glucose,0.5 mg/ml; pH 6.0.

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REDUCTION OF 20-CARBONYL GROUP OF C-21 STEROIDS

presence of this group was confirmed by itsbehavior toward isonicotinic acid hydrazidereagent (26); compound VII gave a yellow spoton TLC plates after 1 to 2 hr. As a final proof,VII was not transformed by strains of B.sphaericus ATCC 7055, 7054, and 245 or by B.lentus ATCC 13805, all known to 1-dehydro-genate various steroids (1, 9, 20, 33), whichindicates that the Al-double bond is alreadypresent in compound VII. The presence of anintact 17,8-methyl-ketone side chain was re-vealed by a positive nitroprusside reaction (13).The 6-hr lag period suggests the presence of

an inducible system. However, the addition of0.05 mg of either peptone, yeast extract, orammonium sulfate per ml to the fermentationmedium as well as the reuse of spores in twosuccessive fermentations did not eliminate thelag period or enhance the yield. Furthermore,the same reaction course was observed whenthe process was conducted in the presence oftetracycline hydrochloride (100 Ag/ml of med-ium), an antibiotic which inhibits protein syn-thesis.Transformation of VI by mycelium of F.

solani. F. solani mycelium transformed VIinto androstadienedione(VIII) and 1-dehydro-testololactone(IX), identified by comparisonwith authentic standards as shown in Table 1.Transformation of 3fl-hydroxy-16a, 17a-

oxido-pregn-5-en-20-one (17a-hydroxy-pregnenolone) by spores and mycelium of F.solani. 3#-Hydroxy-16a, 17a-oxido-pregn-5-en-20-one (17a-hydroxypregnenolone)(XI) wasnot metabolized by either the spores or themycelium.Transformation of compound I and other

steroids by spores or growing cells, or both,of various microorganisms. The activity ofthe spores and mycelia of C. radicicola and S.affinis, as well as the cells of three strains of B.sphaericus and one of B. lentus, on I, VI, andXI and on progesterone(XII) is presented inTable 2.The reaction products listed in Table 2 were

identified on the basis of comparisons of theirRF values and color reactions (isonicotinic acidhydrazide, H2SO0, and nitroprusside tests)with those of authentic samples of the respec-tive steroids.

DISCUSSIONThe spores of F. solani are capable of

bringing about a variety of conversions of thesteroid molecule: introduction of A l-doublebond, reduction of the C-20 carbonyl groupinto a 20a-hydroxy group, cleavage of themethyl ketone side chain, oxygenation of ring

D into a six-membered heterocyclic lactone,and cleavage of the 16a, 17a-epoxide group.

In a previous communication, El-Tayeb etal. (6) suggested that the 20-reductase of C.radicicola (mycelium) is probably competingwith the side-chain-degrading enzymes for C-21 steroids. Thus, substrates which are metab-olized rapidly by side-chain-degrading en-zymes would be transformed very little, if any,into 20-hydroxy compounds, while substrateswhich are slowly metabolized by side-chain-degrading enzymes would be converted into20-hydroxy derivatives. Accordingly, one mayassume that the spores of F. solani would accu-mulate larger quantities of 20-hydroxy steroidsthan the vegetative mycelium, since it hasbeen demonstrated that the former are muchless active in side-chain degradation than thecorresponding growing mycelium (11). FromFig. 2, 4, and 5, it is evident that higher yieldsof II and III accumulated within the first 24 hr,when the spores, instead of the growing cells,of F. solani were incubated with I; further-more, the amount of II produced by thegrowing mycelium disappeared rapidly (after24 hr) from the fermentation medium, whilethe side-chain degradation products (IV and V)accumulated very early. There was also higherrate of side-chain degradation and higheryields of C-19 steroids obtained after 24 hrwith growing mycelium. This suggests compe-tition between side-chain-degrading enzymesand the 20-reductase. It is also interesting tonote that the amount of 20a-hydroxy deriva-tives produced by the mycelium from com-pound I is much higher in F. solani than in C.radicicola (6). This is significant since we knowthat F. solani is a weaker side-chain-degradingfungus than C. radicicola (11).From the results obtained in this study (Fig.

7), it is clear that the course of 20-keto reduc-tion or side-chain degradation, or both, by F.solani (spores or mycelium) was significantlymodified by minor changes in the functionalgroups on the steroid molecule. Thus, whileboth the spores and the mycelium metabolizedprogesterone quantitatively into androsta-diene-dione and 1-dehydrotestololactone (11)without accumulation of any 20-hydroxy com-pounds, the mycelium converted 17a-hydroxy-progesterone into the same products and thespores converted that same steroid into its 1-dehydro analogue without side-chain degrada-tion. Thus, even though 17a-hydroxyprogester-one was not affected by the side-chain-degrad-ing enzymes of the spores, the C-20 keto groupwas not reduced. This inability of the spores toreduce the 20-carbonyl function does not result

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PLOURDE, EL-TAYEB, AND HAFEZ-ZEDAN

TABLE 2. Transformation of various steroids by spores and growing cells of various microorganismsa

Organism and steroid substrate Products Reactions

Cylindrocarpon radicicola ATCC11011;

Septomyxa affinis ATCC 6737(spores, mycelia):

Progesterone, 17a-hydroxy-progesterone

16a, 17a-Oxidopregnenolone16a, 17a-Oxidoprogesterone

Bacillus sphaericus ATCC 7054,ATCC 7055 (vegetative cells):

Progesterone17a-Hydroxyprogesterone16a, 17a-Oxidopregnenolone16a, 17a-Oxidoprogesterone

B. sphaericus ATCC 245,B. lentus ATCC 13805 (vegeta-

tive cells):Progesterone17a-Hydroxyprogesterone16a, 17a-Oxidopregnenolone16a, 17a-Oxidoprogesterone

Bacillus sphaericus ATCC 245,7054, 7055;

B. lentus ATCC 13805 (vegeta-tive cells):

Testosterone"

Androstadienedione

1-Dehydrotestololactone

None20a-Hydroxy derivative20a-Hydroxy-1-dehydro derivative

16a;Hydroxy-1-dehydro-testos-terone

16a-Hydroxy-1-dehydro-testolo-lactone

1-Dehydro derivative1-Dehydro derivativeNone20a-Hydroxy derivative20a-Hydroxy-1-dehydro derivative

1-Dehydro derivative1-Dehydro derivativeNone1-Dehydro derivative

Androstenedione

1-Dehydrogenation, side-chaindegradation

1-Dehydrogenation, side-chaindegradation, ring D lactoniza-tion

NilReduction of the C-20 ketoneReduction of the C-20 ketone, 1-

dehydrogenation1-Dehydrogenation, side-chain

degradation, epoxide cleavage1-Dehydrogenation, side-chain

degradation, epoxide cleavage,ring D lactonization

1-Dehydrogenation1-DehydrogenationNilReduction of the C-20 ketoneReduction of the C-20 ketone, 1-dehydrogenation

1-Dehydrogenation1-DehydrogenationNil1-Dehydrogenation

Oxidationgroup

of the 17fl-hydroxy

aSpores, 1.5 x 108/ml; substrate, 0.5 mg/ml; glucose, 0.5 mg/ml; incubation, 24 to 72 hr." Incubation period, 24 hr.

from impermeability, since the substrate couldbe transformed into its 1-dehydro derivative. Al-ternative explanations present themselves forthe cleavage of the side chain of 17a-hydroxy-progesterone by mycelium and not by spores.(i) The spores may contain only 1-dehydro-genase and lack the side-chain-degrading en-zymes. (ii) The main reaction in both myce-lium and spore processes may be 1-dehydro-genation; further incubation may induce denovo protein synthesis of the side-chain-cleaving enzymes in the mycelium. This in-duction would not take place in spores due to

absence of adequate nitrogen supplement. (iii)Both mycelium and spores contain both 1-dehydrogenase and side-chain-degrading en-zymes. In spore-mediated reactions, the side-chain-degrading enzymes may be inactive dueto a lack of certain cofactors which may bepresent in the mycelium-mediated reactions.The preparation of a cell-free extract fromspores may explain the difference of activity.Vischer and Wettstein (32) already found thatthe presence of a 17a-hydroxy group on thesteroid molecule blocks side-chain degradationby the F. solani mycelium; however, our obser-

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REDUCTION OF 20-CARBONYL GROUP OF C-21 STEROIDS

+

CH3a-0

0 oH

-OH -OH

SIMV

¶H3

Ho-c-H

o0" lZ

S'M

FIG. 7. Transformation of steroids by the spores and the mycelium of Fusarium solani. S, spores; M, my-celium.

vation is the first example of such a differencebetween two different forms (spores and myce-lium) of a given strain.The question as to whether a nonspecific 17#-

hydroxy steroid dehydrogenase might be re-sponsible for catalyzing the reduction of theketo function at C20 has been investigated.Although all examined strains of B. sphaericusand B. lentus (Table 2) oxidized the 17ft-hy-droxy group of testosterone, only two strains ofB. sphaericus (ATCC 7054, 7055) were able toreduce the C20 ketone of 16a, 17a-oxidopro-gesterone. This observation attenuates the pos-

sible participation of a 17fl-hydroxy steroiddehydrogenase.To examine the need for a 16a,17a-oxido

group as the only requirement for the activityof the 20-reductase system, 16a, 17a-oxido-pregnenolone was used as a substrate. Neitherthe mycelium nor the spores of F. solani couldtransform it. However, the permeability phe-nomena are not excluded.The ability of several microbial cultures to

reduce the 20-carbonyl group of 16a, 17a-oxi-doprogesterone to the 20a-hydroxy group andthe influence of structure alterations in the

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PLOURDE, EL-TAYEB, AND HAFEZ-ZEDAN

molecule on their activity are illustrated inTable 2. It is probable that the C20 ketone re-duction or the side-chain degradation, or both,could be hindered through alteration of thespatial relationship in the steroid molecule insuch a way that the points of reactivity be-tween the enzyme and the substrate may beblocked.The possibility of a 20-hydroxy intermediate

in steroid side-chain degradation was reportedby Sweat and co-workers (27), who proposedthat 17a, 20a-dihydroxypregn-4-en-3-one canserve as an intermediate precursor of andro-stenedione in bovine and human ovaries, whileSebek et al. (22) suggested the possibility ofparticipation of these 20-dihydro compoundsas intermediates in microbiological side-chaindegradation. Our results showed that 20a-hy-droxy-16a, 17a-oxidopregn-4-en-3-one wasmetabolized at a slower rate than 16a, 17a-oxidoprogesterone and the 20-reductase ofother microorganisms which do not carry outside-chain cleavage (Table 2). It is thereforeunlikely that 20a-hydroxy compound is anobligatory intermediate in the side-chain deg-

C H3C=O

radation by F. solani spores. This has also beenshown to be true with C. radicicola mycelium(6).Rahim and Sih reported that 20a-hydroxy-

16a, 17a-oxidopregn-4-en-3-one was not me-tabolized by the side-chain-degrading enzymesisolated from mycelium-free extracts of C. rad-icicola (19). Similar observations were reportedfor other biological systems; Lynn and Brown(14) have demonstrated that testicular micro-somes failed to cleave the side chains of 20a-and 20#-hydroxysteroids, and Ball and Kadis(2) reported that the side chain of progesteronewas not cleaved with sow ovary, although a20a-hydroxy compound (17a, 20a-dihydroxy-pregn-4-en-3-one) was formed. Therefore, itappears that these 20a-hydroxy steroids haveno biological role in side-chain degradationand that they are unusual metabolites of 16a,17a-oxidoprogesterone.Figure 8 represents three alternate mecha-

nisms for the opening of the 16a, 17a-oxidogroup. Mechanism A involves the opening ofthe oxido group into a 17a-hydroxy group (thepresence of a 17A3-side chain prevents the at-

CH3C=O-OH

A

[ H ]

0I01

C H3C=0

B

\ H I]C

C H3

C=O--OH

0 0rj 11III*O-C-R O-C -CH3

-0

H.

+ R-C-0-

2°

0+ C H3- C -OH

FIG. 8. Possible mechanisms for the cleavage of the 16a, 17a-oxido group of 16a, 17a-oxidoprogesterone.

'j CH3H -0-C -0-

--0

I--,0

0

-OH

610 APPL. MICROBIOL.

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REDUCTION OF 20-CARBONYL GROUP OF C-21 STEROIDS

tack of the hydride ion from the back side ofthe molecule, thereby preventing the forma-tion of a 16a-hydroxy derivative) via a reduc-tive type of opening in a manner similar tothat by metal hydrides. Side-chain degrada-tion of the resulting 17a-hydroxy compoundwould yield the corresponding 17-ketosteroidwhich on reduction gives the 17fl-hydroxy de-rivative and on oxidation gives the respectivesteroid lactone. The presence of a 16a-hydroxygroup on the molecules of the side-chain deg-radation metabolites (IV and V) and the ina-bility of the spores to cleave the side chain of17a-hydroxyprogesterone rule out this mecha-nism. Mechanism B could proceed via theopening of the 16a, 17a-oxido group into a 16a,17a-dihydroxy derivative which parallels theacid-catalyzed opening of epoxides. Side-chaindegradation followed by reduction or oxygena-tion would give rise to 16a, 17#-dihydroxy-and 16a-hydroxy ring D lactone-steroids, re-spectively. This mechanism is also ruled outby the fact that the presence of a 17a-hydroxyfunction in the steroid structure inhibited theside-chain degradation activity of the spores.Mechanism C could involve side-chain degra-dation of the 16a, 17a-oxido compound fol-lowed by a nonenzymic rearrangement to the16a-hydroxy-17-ketone. Reduction of the 17-keto group would give the 16a, 17fl-dihydroxymetabolite (V), while its oxidation through aBaeyer-Villiger-type reaction could yield thecorresponding lactone (IV). Our results are incomplete agreement with mechanism C, whichinvolves a nonenzymic epoxide opening.The resistance of 16a-hydroxy-1-dehydro-

testosterone (V) to further oxidation is con-sistent with the observation of Talalay andMarcus (28) that 17f3-hydroxysteroid dehydro-genase from Pseudomonas testosteroni wasunable to oxidize 17fl-hydroxy steroids bearingoxygen functions at C, and with the observa-tion of El-Tayeb et al. (6), who reported thatV is a final product in the fermentation of16a,17a-oxidoprogesterone with C. radicicola(mycelium). However, the reverse reaction canobviously take place, since 16a-hydroxy-an-drostenedione was readily transformed to V bynongerminating spores and mycelium of F. so-lani as indicated by X - V on Fig. 7.We conclude that the metabolism of 16a,

17a-oxidoprogesterone by nongerminatingspores of F. solani can tentatively be repre-sented by Fig. 9, which is consistent with previ-ous findings (6) with C. radicicola mycelium.The 6-hr lag period that was apparent in the

bioconversion of 17a-hydroxyprogesterone byspores of F. solani (Fig. 6) suggested that en-zyme adaptation had occurred. As described

CH3C:C

cIllz) H 0-- I

)~~~~~HO-C -H

III0

)I- CH3 OH

-H O

0-OH

/ o -OH

o IV

FIG. 9. A proposed mechanism for the metabo-lism of 16a,177a-oxidoprogesterone by the spores ofFusarium solani.

by Mandel and Vitols (15) for the induced tre-halose mechanism in spores of the fungusMyrothecium verrucaria, the de novo proteinsynthesis could occur at the expense of theendogenous amino acid pool of the spores. Weexamined this possibility by three independantmethods: (i) reuse of one batch of spores insuccessive fermentations of the same sub-strate; (ii) addition of a very low concentrationof an exogenous protein supplement (0.05 mgof peptone, yeast extract, or ammonium sul-fate per ml of medium) to the reaction me-dium before incubation; and (iii) biotransfor-mation in the presence of a protein synthesisinhibitor (100 jig of tetracycline HCl per ml ofmedium).

In all cases, the 6-hr lag period and the samepattern of transformation were obtained, thuseliminating the possibility of enzymatic adap-

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612 PLOURDE, EL-TAYEB, AND HAFEZ-ZEDAN

tation during the process (although one has tobear in mind the question of cell permeabilityto the antibiotic used). This observation, whichis consistent with the report of Vezina et al.(30) on hydroxylation of steroids by the conidiaof Aspergillus ochraceus, is being investigated.

ACKNOWLEDGMENTS

We wish to express our sincere appreciation to ClaudeVezina, Director of the Department of Microbiology, Labor-atoires de Recherche Ayerst, Montreal, for critical reading ofthe manuscript and for valuable suggestions.

H. H. Zedan wishes to express his profound gratitude toProf. Dr. Julien Braun, Dean of Facult6 de Pharmacie,Universit6 de Montr6al, for sustained encouragement duringmost of this work and to the Department of Microbiology,Faculty of Pharmacy, University of Cairo, Cairo, Egypt,where this work was initiated.We are indebted to the Canadian Foundation for the

Advancement of Pharmacy for their financial support of thisproject.

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2. Ball, J. H., and B. Kadis. 1964. Steroid hydroxylations.I. Biosynthesis of 17a,20a-dihydroxy-pregn-4-en-3-one by sow ovary. Steroids 4:533-538.

3. Camerino, B., and R. Sciaky. 1959. Azione del lievitofermentante sul 4fj, 5-epossi-progesterone. Gazz.Chim. Ital. 89:654-662.

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12. Hafez-Zedan, H., and R. Plourde. 1971. "Spore platemethod" for transformation of steroids by fungalspores entrapped in silica gel G. Appl. Microbiol. 21:815-819.

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14. Lynn, W. C., and R. H. Brown. 1958. The conversion ofprogesterone to androgens by testes. J. Biol. Chem.232:1015-1030.

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20. Raspe, G., K. Kieslich, E. Oliver, R. Muller, and B.Wagner. 1967. p. 308-309. In W. Charney and H. L.Herzog (ed.). Microbial transformations of steroids.Academic Press Inc., New York and London.

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22. Sebek, 0. K., L. M. Reineke, and D. H. Peterson. 1962.Intermediates in the metabolism of steroids by Peni-cillium likacinum. J. Bacteriol. 83:1327-1331.

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25. Singh, K., S. N. Sehgal, and C. V6zina. 1967. Transfor-mation of steroids by Mucor griseo-cyanus. Can. J.Microbiol. 13:1271-1281.

26. Smith, L. L., and T. Foell. 1959. Differentiation of A4-3-ketosteroids and A '4-3-ketosteroids with isonicotinicacid hydrazide. Anal. Chem. 31:102-105.

27. Sweat, M. L., D. L. Berliner, M. J. Bryson, C. Nabors,J. Haskell, and E. G. Holmstrom. 1960. The synthesisand metabolsim of progesterone in human and bovineovary. Biochim. Biophys. Acta 40:289-296.

28. Talalay, P., and P. I. Marcus. 1956. Specificity, kinetics,and inhibition of a- and ,B-hydroxysteroid dehydro-genases. J. Biol. Chem. 218:675-690.

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30. Vezina, C., S. N. Sehgal, and K. Singh. 1963. Transfor-mation of steroids by spores of microorganisms. I.Hydroxylation of progesterone by conidia of Asper-gillus ochraceus. Appl. Microbiol. 11:50-57.

31. Vezina, C., S. N. Sehgal, and K. Singh. 1968. Transfor-mation of organic compounds by fungal spores. Adv.Appl. Microbiol. 10:241.

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