Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius

356

Biochirniea et Biophysica Acta 881 (1986) 356-363

Elsevier BBA 22316

Characterization and partial purification of s q u a l e n e - h o p e n e cyclase from B a c i l l u s a c i d o c a i d a r i u s Brigitte Seckler and Karl Poralla * Institute for Biology 11, Microbiology 1, UniversiO' of 7~fibingen, A uf der Morgenstelle 28. D - 7400 Tfibingen ( F R. G.)

(ReceivedOctober 30th, 1985) (Revised manuscript receivedJanuary 31st, 1986)

~ey words: Hopanoid synthesis; Squalenecyclization;Thermophilicenzyme; (B. acidocaldarius)

A membrane-bound enzyme activity from Bacillus acidocaldarius converted squalene to two pentacyclic triterpenes, hop-22(29)-ene and hopan-22-ol. The products were formed in a constant molar ratio of hopene: hopanol, 5: 1, probably through parallel, and not successive, reactions. The conversion was independent of oxygen, in contrast to the biosynthesis of sterols from epoxysqualene in eukaryotes. The squalene-hopene cyclase was purified 270-fold by extraction from B. acidocaidarius membranes at low concentrations of Triton X-100 followed by DEAE-cellulose chromatography. The enzyme showed optimal rates of squalene conversion at pH 6 and 60°C, corresponding to the intracellular pH and the optimal growth temperature of the bacterium. The apparent K m for squalene is 3 #M. Effective inhibitors of the enzyme were some sulfhydryl reagents and the histidyl reagent diethyl pyrocarbonate. The squalene-hopene cyclase, like several eukaryotic epoxysqualene cyclases, was strongly inhibited by AMO 1618 and by high ionic strength. On the basis of these and other similarities a phyiogenetic relationship between the key enzymes of steroid and hopanoid biosynthesis was envisaged.

Introduction While most eukaryotic organisms contain sterols, mainly to stabilize their membranes [1,2], sterols occur in only a few bacteria [1]. However, many prokaryotes synthesize hopanoids, a class of pentacyclic triterpenoids [3]. Hopanoids influence lipid order, membrane permeability, and fluidity of artificial membranes in a manner very similar to sterols [4-7] and substitute for sterols in stimulation of M y c o p l a s m a growth [8]. Thus, hopanoids may fulfil a role in bacterial membranes similar to that of sterols in eukaryotes. Squalene is a common intermediate in the bio* To whom correspondenceshould be addressed. Abbreviation: AMO 1618, (2-isopropyl-5-methyl-4-trimethylammonium chloride)phenyl-l-piperidinecarboxylate.

synthesis of both classes of triterpenoids. While sterols are synthesized by cyclization of epoxysqualene formed from squalene by O2-dependent oxidation, in the biosynthesis of hopanoids squalene is cyclized without prior epoxidation [1,9,10]. As hopanoids have been proposed to be phylogenetic precursors of sterols [11,12], the epoxysqualene-sterol cyclases may have evolved from squalene-cyclizing enzymes. Elucidation of this possible relatedness requires characterization of both classes of enzymes. Whereas several of the eukaryotic, epoxysqualene-dependent enzymes are well investigated [13-15], examination of squalene-cyclizing activities in bacterial cell homogenates has been limited to the characterization of substrate specificity and catalytic mechanism [16-181. In this paper we present the first partial purifi-

0304-4165/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

357 cation and characterization of a squalene-hopene cyclase. The enzyme was derived from Bacillus acidocaldarius, a thermoacidophilic bacterium naturally occurring in hot springs [19] and containing various hopanoids [20].

Pooled fractions containing squalene cyclase activity were concentrated by ultrafiltration (Amicon M 12 with membrane filter YM 5), and detergent was removed by batch chromatography on Servachrom XAD 2 [21].

Materials and Methods

Enzyme assay Unless otherwise indicated, the enzyme-containing fractions and an emulsion of the substrate (100 /~1 500 /~M [4,8,12,17,21-3H]squalene, 37 MBq/mmol, New England Nuclear, Dreieich, F.R.G., in 10 mM sodium citrate (pH 6) plus 1 m g / m l Triton X-100) were incubated for 1 h at 60°C in incubation buffer (100 mM sodium citrate (pH 6)) in a total volume of 1 ml. To obtain the above-mentioned specific activity the tritiated squalene (147-733 GBq/mmol) was diluted with unlabeled squalene (Sigma). The purity of the unlabeled squalene was measured as 98% in our laboratory by gas-liquid chromatography. The tritiated squalene contained no radioactive impurities according to scanning of a thin-layer chromatogram. The enzyme reaction was started by addition of the substrate emulsion to the reaction mixture preincubated for 5 min at 60°C and was stopped by extraction of substrate and products with 2 vol. of n-hexane/isopropanol (3:2, v/v) [22]. The sample was concentrated by evaporation of the solvent and subjected to thin-layer chromatography after the addition of 10 /~g each of carrier squalene, hop-22(29)-ene and hopan-22-ol. The thin-layer chromatograms (Silica gel 60, Merck, Darmstadt, F.R.G.) were developed twice: first 6 cm in CHC13 and then 18 cm in n-hexane. Substrate and products migrated with relative mobilities of R v = 0 . 4 , 0.6 and 0.1 for squalene, hop-22(29)-ene and hopan-22-ol, respectively. The compounds were visualized very sensitively by excitation at ?~= 360 nm of berberine chloride (10 ffg/ml in 95% ethanol) [3]. The fluorescent spots of substrate and products obtained after drying were carefully scraped from the chromatograms and radioactivity was quantified after suspending by liquid scintillation counting. The quench was always at 5-10% as measured by external standardization. For the enzyme assay always double determinations were run. In most incubations less than 20% of the squalene was converted to products.

Bacterial strain and growth conditions B. acidocaldarius, strain 104-IA, obtained from Dr. T.D. Brock, University of Wisconsin, Madison, U.S.A., was grown in sporulation medium [19] at pH 3.5 and 50°C. This temperature is about 10°C below the temperature optimum for growth. The values were for agitation 300 rev./min and for aeration 250 1/h in a 20 1 fermentation vessel (model C6 from Biolafitte, France). Logarithmically growing cells were harvested and washed twice in incubation buffer or buffers used for membrane preparation. Membrane preparation Harvested cells were resuspended in 100 mM sodium citrate, pH 6 (100 mg wet weight per ml) and sonicated at O°C for 9-10 min in 3 min intervals at a power uptake of 45 W (Branson Sonifier B 12). After removing residual cells by low-speed centrifugation, membranes were sedimented at 105000 Xg for 1.5-2 h at 4°C and washed two or three times with 10 mM sodium citrate (pH 6). For enzyme purification, 10 mM Tris-HC1 (pH 8) was present during the disruption of cells. Enzyme purification Sedimented membranes were resuspended in 10 mM sodium citrate (pH 6) (12 mg membrane protein/ml) at room temperature and mixed rapidly with an equal volume of 3.5 m g / m l Triton X-100 (Serva, Heidelberg, F.R.G.) in this same buffer. The mixture was incubated for 30 min at 60°C and then centrifuged at 105 000 X g for 1.5 h at 18°C. The supernatant was adjusted to pH 6.5 with NaOH and chromatographed on a DEAEcellulose column (Servacel 23 SH; column volume 0.5 ml per mg of applied protein). The column was washed with 2 vol. of 1 m g / m l Triton X-100 in 10 mM sodium citrate (pH 6.5). Then the enzyme activity was eluted with a linear gradient of 0-0.2 M NaC1 in the same buffer over 4 column volumes.

358

The products of the enzymatic reaction were identified by GC-MS at the Institute of Organic Chemistry of the University of Ti~bingen. They were separated on a 30 m SE54 glass-capillary column in the gas chromatograph Spectrovap 2900 (Carlo Erba) at a temperature regimen of 5 K / r a i n from 20 to 150°C and of 10 K / m i n from 150 to 300°C. After splitting into a mass spectrograph (Varian Mat 1125) the probe was ionized at 80 eV.

Protein determination Protein was determined by the method of Lowry et al. modified as described by Peterson [23] using bovine serum albumin as standard.

= 410 for hopene and M+/e = 428 for hopanol) and fragmentation pattern ( m / e = 370, 367, 207 and 149). These values agree well with those obtained for the authentic substances [24,25]. The products were formed in a molar ratio of 1:5 hopanol/hopene, independent of variations in pH (pH 3-8) and temperature (30-70°C). The same ratio of products was obtained when Tween 80 or sodium deoxycholate were used instead of Triton X-100 for squalene emulsification. The formation of hopene and hopanol is apparently due to a single enzyme, as no separation of hopene- and hopanol-forming activity nor alteration of the ratio of products formed was observed during enzyme purification.

Results

Conversion of squalene by B. acidocaldarius Sonicated B. acidocaldarius cells convert [3H]squalene to two major products. More than 95% of the original radioactivity was found in squalene and these products. Substrate and products could be extracted by organic solvents and the chromatographic mobilities of the products were unaffected by treatment with 5% K O H in methanol. Therefore, the two new compounds are nonesterified derivatives of squalene. The products co-migrate with hopan-22-ol and hop-22(29)-ene in thin-layer and gas chromatography. The identities of both compounds were confirmed by mass spectrometry in terms of molecular weight ( M + / e

Subcellular location and partml purification of the enzyme The squalene-cyclizing activity was located in the crude membrane fraction on the basis of its cosedimentation with the membrane marker succinate dehydrogenase. Neither variation of pH between pH 6 and 9 or ionic strength (distilled H 2 0 to 1 M KCI/0.1 M potassium phosphate (pH 7.4) nor addition of the chelating agent EDTA (0.1 M) resulted in the release of significant squalene cyclase activity into the supernatant. Detergents were more effective. Sodium deoxycholate, 1 m g / m l at a protein concentration of 2 mg/ml, solubilized 76% of squalene cyclase activity, but its use was limited to

TABLE 1 P A R T I A L P U R I F I C A T I O N O F T H E S Q U A L E N E - H O P E N E CYCLASE F R O M B A C I L L U S A C I D O C A L D A R 1 U S Values are normalized from a purification starting with 2.5 g of total cell protein and an enzyme activity of 32.2 /~mol s q u a l e n e / h in the cell free homogenate. Purification step

Fraction

Total protein

Squalene cyclase activity

cell-free homogenate

100

100

Purification factor 1

Membrane preparation

supernatant pellet

74.1 18.4

1 85

Solubilization

supernatant pellet

2.1 16.5

103 8

49

DEAE-chromatography

combined fractions, after removal of detergent

33

270

0.12

4.5

359 B

i

i

,

1

a4

>

,o

< z~

/

> Z

t' I

w

IO0

2OO

Fig. 1. Chromatography of squalene-hopene cyclase on DEAE-cellulose in the presence of Triton X-100. The supernatant obtained by Triton X-100 extraction of B. acidocaldarius membranes (50 mg of protein) was applied to a 20 ml DEAEcellulose column and proteins were eluted. The protein concentration ( - . . . . . . A), conductivity (e . . . . . . e), and squalene cyclase activity (© (3) of the fractions were monitored.

near neutral pH. Starting at a detergent:protein ratio of 0.2-0.3 (w/w), Triton X-100 solubilized about 95% of the squalene cyclase activity accom-

0

10

40

...........

• 2

70

(o()

~ , 4

i 6

I 8

pH

Fig. 3. Temperature- (A) and pH- (B) dependence of squalene cyclase activity in B. acidocaldarius ultrasonic homogenate. Squalene cyclase activity was measured with modifications given below, and was normalized to the activity maximum observed (0.85 (A) and 3.9 (B) n m o l / h per mg protein, respectively. (A) Incubation at varying temperature for 45 rain with 4.5 mg protein/assay in potassium phosphate buffer (pH 7.4). (B) Incubation at 60°C and varying pH for 17 h with 0.14 mg protein/assay in potassium-citrate buffer. Identical pH optima were observed in sodium-citrate, potassium-phosphate, sodium-citrate/phosphate and potassium/Tris-phosphate buffers.

12

Mr

108000

75

o

000 O

50 000 45 000

E

36 000

4

1

2

3

4

Fig. 2. Purification of squalene cyclase followed by gel electrophoresis. Samples (40/~g of protein) of B. acidocaldarius membranes (lane 1), residual membranes after extraction with Triton X-100 (lane 2), the detergent extract (lane 3), and of the combined fractions after DEAE-cellulose chromatography (lane 4) were separated on a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate [26]. Protein was stained with Serva Blue R 250 (Serva).

2

4

Fig. 4. Eadie-Hofstee plot [27] of initial velocities of squalene cyclization at varying substrate concentrations. B. acidocaldarius membranes were extracted with Triton X-100 and, after removal of the detergent, squalene cyclase was assayed in the extract (28 p~g protein/assay) for 30 s - 3 h at squalene concentrations of 20 mM-200 #M. The apparent K m was 3 ~tM.

360 TABLE II ~O

EFFECT OF VARIOUS REAGENTS ON SQUALENE CYCLASEACTIVITYFROM B. ACIDOCALDAR1US

E E c

Reagent 2c > i-

p-Chloromercuribenzenesulfonic acid ~

,,J

Final concen- Inhibitionof squatration (mM) lene cyclase(%) 1 96

N-Ethylmaleimide b

1 5

20 65

=,1

Diethyl pyrocarbonate b

0.1 5

81 92

0

AMO 1618 b

0.001 0.01 1

78 92 99

1

0

v

1£

0.5

1

,,~, (M/ Fig. 5. Effectof ionic strength on squalene cyclaseactivity from B. acidocaldarius. Ultrasonic homogenate(0.28 mg protein/as-

say) was assayed for squalene cyclase activity by standard enzyme assays in 10 mM (e e) and 100 mM (O O) potassium phosphate buffer (pH 7.4) for 3 h. panied by liberation of only 15-20% of total membrane protein. Raising the pH from 6 to 8 and lowering the ionic strength resulted in the release of about 75% of the crude membrane protein, whereas the enzyme activity was quantitatively retained in the membrane fraction. Therefore, for preparation of membranes used for enzyme purification, cells were disrupted in the presence of 10 mM Tris-HC1 (pH 8). Compared to the cell-free homogenate, squalene cyclase activity was enriched about 5-fold in membranes prepared at this pH. Extraction with Triton X-100 (1.75 m g / m l at a protein concentration of 6 m g / m l ) quantitatively solubilized the enzyme activity but released only about 10% of the total membrane protein. An overall nearly 50-fold purification was achieved by these two steps. After removal of detergent, the solubilized enzyme was stable for at least 2 weeks at 4°C without significant loss of enzyme activity. Further purification of the enzyme could be achieved by chromatography on DEAE-cellulose in the presence of detergent (Fig. 1). The yield of the enzyme from this procedure was low and resulted in a 6-fold enrichment for this step, repre-

Phenylmethylsulfonyl fluoride b

Assayed using ultrasonic homogenate of B. acidocaldarius cells (0.9 mg protein/assay). b Assayed using the detergent-depleted Triton X-100 extract from B. acidocaldarius membranes(15-30 ~g protein/assay).

a

senting a 270-fold overall purification from the ultrasonic homogenate. Table I summarizes all steps of the purification procedure. The protein purification was followed by gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 2). Characterization o f the enzyme

Several characteristics of the squalene-cyclizing activity were investigated. The enzymatic conversion of squalene was independent of the presence of oxygen, as 105% of the aerobic cyclization rate was obtained when the reaction is performed in a solution saturated with N 2. The squalene cyclase showed optimal conversion rates at 60°C and pH 6 (Fig. 3). The apparent K m determined by kinetic studies with varying squalene concentrations was 3 /~M (Fig. 4). Thus, the purified enzyme exhibited a high affinity for the substrate squalene. High ionic strength inhibited the enzyme significantly (Fig. 5, and like most epoxysqualene cyclases squalenehopene cyclase from B. acidocaldarius was strongly inhibited by AMO 1618 (Table II). Chemical modification identified two classes of residues of possible catalytic significance. The water-soluble organic mercurial p-chloromercuri-

361

benzenesulfonic acid virtually completely inhibited the enzyme (Table II). The more lipophilic sulfhydryl reagent N-ethylmaleimide evoked only partial inhibition. Therefore, a cysteine residue more accessible from the aqueous than from the lipid or detergent phase may be of catalytic significance. Diethylpyrocarbonate evoked nearly complete inhibition (Table II), suggesting the possible participation of a histidine residue in catalysis. The pH optimum near 6 may also reflect the importance of histidine residues. Lastly, the failure of phenylmethylsulfonyl fluoride to inhibit the enzyme makes the involvement of a serine residue unlikely. Discussion

Hop-22(29)-ene and hopan-22-ol were formed from squalene by an enzyme activity present in B. acidocaldarius cell homogenate. Identical products have been found in Acetobacter species [16,17] and in Methylococcus capsulatus [18]. No further conversion of the two products is detected under the reaction conditions, probably due to the lack of an energy-rich second substrate, possibly phosphoribosyl pyrophosphate [28]. Both products are formed in a constant molar ratio, irrespective of reaction conditions and degree of enzyme purification. Thus, a single, enzyme-catalyzed process appears most likely. These products could arise by either alternate or sequential reaction pathways starting from a carbonium intermediate during the H+-initiated cyclization of squalene (Fig. 6). In the first case, attack of OH- on the carbonium ion could compete with the loss of H + and formation of the 22,29 double bond yielding hopene. In the sequential pathway, hopene could arise by dehydration of hopanol. We favor the former mechanism, because in preliminary experiments no conversion of hopanol to hopene or hopene to hopanol could be demonstrated, and no formation of squalene from either hopene or hopanol was observed (data not shown), Moreover, the different ratios of reaction products observed in this study (hopene : hopanol, 5:1) and by Rohmer et al. (hopene:hopanol = 1 : 1, Ref. 17) argue against enzyme-catalyzed equilibrium between hopene and hopanol. As hopene- and not hopanol- is the biosynthetic

precursor of bacteriohopane tetrol (1,2,3,4-tetrahydroxypentane-29-hopane), hopanol is proposed to be formed by a side-reaction of squalene-hopene cyclase, probably favored under conditions in vitro. Squalene cyclase from B. acidocaldarius is a membrane-bound enzyme, as are the squalene-tetrahymanol cyclase from Tetrahymena pyriformis [29] and the microsomal epoxysqualene cyclases from hog liver [13], pea seedlings [13] and Ochromonas malhamensis [14], w h e r e a s oxidosqualene-lanosterol cyclase from yeast [30] and the dioxidosqualene-c~-onocerine cyclase from Ononis spinosa [31] do not cosediment with membrane fractions. Squalene-hopene cyclase from B. acidocaldarius could be released from the particulate fraction by detergent extraction, whereas no release of enzyme activity is observed under conditions reported to result in the solubilization of membrane-associated proteins [32]. Sodium deoxycholate, which has been used to solubilize oxidosqualene-cyclizing enzymes [13,14], also solubilized large amounts of squalene-hopene cyclase at detergent concentrations comparable to those used for the solubilization of the eukaryotic enzymes [13]. Because of the low solubility of sodium deoxycholate at the pHoptimum of the enzyme, the solubilization by Triton X-100 was more favorable. Optimal solubilization was achieved at detergent concentrations (0.2-0.3 mg Triton X-100 per mg of membrane protein) lower than those reported to disrupt membrane structure (1-2 mg Triton X-100 per mg of membrane protein, cf. Ref. 33). Therefore, we suppose that squalene-hopene cyclase is anchored in the membrane by strong hydrophobic interactions but is not an integral membrane protein. The squalene-hopene cyclase from B, acidocaldarius could be purified 270-fold compared to the cell free homogenate by detergent extraction of cytoplasmic membranes followed by DEAE-cellulose chromatography in the presence of detergent. Preparation of membranes and solubilization of the enzyme resulted in an overall 50-fold purification, whereas comparable purification of epoxysqualene cyclases (12- to 20-fold) could be achieved only after ammonium sulfate precipitation of the enzyme solubilized by sodium deoxycholate treatment of microsomes [12,13]. Although more than 90% of the protein was sep-

362

SQUALENE

4

HOPAN-22

-OL

HOP-22

(29)- ENE

Fig. 6. Proposed reaction mechanism for the proton-catalyzed cyclization of squalene to hop-22(29)-ene and hopan-22-ol occurring in parallel via the hopenyl cation.

arated from squalene-cyclizing activity by chromatography on DEAE-cellulose, only a net 5-fold purification was achieved in terms of the specific activity, probably due to enzyme inactivation during chromatography. Electrophoresis of the fractions after different purification steps disclosed a significant enrichment of a protein of M r 75 000, which may represent the enzyme examined here. This apparent molecular weight is comparable to the M r = 90000 of epoxysqualene-lanosterol cyclase from hog liver [15]. With a K m of 3 I~M the squalene-hopene cyclase shows a significantly higher affinity for its substrate compared to epoxysqualene cyclizing enzymes from O. malharnensis and hog liver (Kin = 100/~M and 25 #M, cf. Refs. 14,15). The specific activity of 60 nmol squalene converted/mg membrane protein per h in the membrane fraction is comparable to that for epoxysqualene-cyclizing activities reported for microsomal fractions from O. malhamensis, hog liver, and hog brain with 12 nmol, 4-47 nmol, and 20 nmol of epoxysqualene converted/mg per h, respectively [10]. Like the epoxysqualene-lanosterol cyclase from yeast [30],

squalene-hopene cyclase from B. acidocaldarius is inhibited by high ionic strength, in contrast to comparable enzymes from O. malhamensis [14], hog liver [30] or O. spinosa [31]. The pH and temperature optima of the enzyme from B. acidocaldarius (pH 6 and 60°C) correspond to the temperature optimum for growth of the bacterium [19] and with the reported intracellular pH [34]. The observed inhibition of squalene-hopene cyclase by the thiol reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide indicates the presence of an essential SH group in the enzyme. An essential thiol group has also been suggested for epoxysqualene cyclases from O. malhamensis [13] and hog liver [32]. The imidazole reagent diethyl pyrocarbonate also inhibited the cyclase. This may suggest an essential histidine residue. As in the cases of the epoxysqualenecycloartenol cyclase in tobacco plants and Digitalis and of the epoxysqualene-lanosterol cyclase from rat liver [35], the bacterial squalene-hopene cyclase is very strongly inhibited by AMO 1618. Squalene-hopene cyclase from B. acidocaldarius thus shares several properties with epoxysqualene cyclizing enzymes from eukaryotes, although its natural substrate is the non-oxygenated squalene. Basically, procaryotic squalene cyclases may also use epoxysqualene as a substrate. For example, the squalene cyclizing activity in Acetobacter cell homogenates has been shown to recognize and cyclize both stereoisomers (R and S) of 2,3oxidosqualene, in addition to its natural substrate, squalene [16,17]. These results indicate a close relationship between squalene- and oxidosqualene-cyclizing enzymes. Further insight into the evolutionary connection between these two classes of enzymes may be achieved when nucleotide or amino-acid sequences are available for comparison. The partial purification of the squalene-hopene cyclase from B. acidocaldarius reported here may be a step on this way.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (grant Po 117/8-1). We thank Dr. J.K. Wright for help with the preparation of the manuscript.

363

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