Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

ELSEVIER

FEMS Microbiology

Letters 127 (1995) 151-155

Purification and characterization of Saminolevulinic dehydratase from Methanosarcina barkeri Suresh Bhosale

*, Deepa

acid

Kshirsagar, Prashant Pawar, Tulsir’am Yeole, Dilip Ranade

Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India Received 19 November 1994; revised 25 January 1995; accepted 27 January

1995

Abstract S-Aminolevulinic acid dehydratase from the archaebacterium Methanosurcina barkeri resembles the mammalian and yeast enzymes in its activation by Zn2+, whereas its activation by K+ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni2+ which is a component of F4so, a cofactor present mainly in methanogens. The M, of 280 000 for the native enzyme and 30 000 f 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of S-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates. Keywords: Archaebacterium;

Methanosarcina

barkeri; SAminolevulinic

1. Introduction Strictly anaerobic, methane-producing phenotypes of archaebacteria contain two major tetrapyrroles: (i) factor Fd3,,, a nickel tetrapyrrole; and (ii) 5-hydroxybenzimidazolyl cobamide, factor III [l]. All naturally found tetrapyrroles are derived from porphobilinogen (PBG), which is formed from two molecules of 5aminolevulinic acid (ALA) by the catalytic activity of 5aminolevulinic acid dehydratase (ALA dehydratase) [2]. This enzyme was first purified from bovine liver by Gibson et al. [3]; since then there have been numerous reports on its isolation and characterization from various sources. Relatively lit-

* Corresponding author. Tel: +91 Fax: + 91 (212) 362972.

(212) 344357

Federation of European Microbiological SSDI 0378-1097(95)00057-7

Societies.

and 343683;

acid dehydratase

tle information is available on ALA dehydratase of methanogens. Relation of this enzyme with synthesis of factor F430 and 5hydroxybenzimidazolyl cobamide has been reported 141. We have isolated ALA dehydratase from Methanosarcina barkeri and observe that the enzyme has some properties of eucaryotic ALA dehydratase and few characteristics of procaryotic ALA dehydratase.

2. Materials and methods 2.1. Chemicals and gases 5Arninolevulinic acid, DNase and protamine sulphate were purchased from Sigma Chemical Company, USA. Chemicals for column chromatography

S. Bhosale et al. / FEMS Microbiology Letters 127 (1995) 151-155

152

were from Pharmacia LKB Biotechnology, Sweden. Nitrogen and hydrogen gases were of IOLAR grade. 2.2. Cultivation of organism and preparation

of cell

extract

Cells of Methanosarcina barkeri (MCM B-707, obtained from the National Culture Collection of Methanogens, MACS, Pune, India) were grown in a basal medium containing 100 mM methanol under nitrogen atmosphere at 37” C to obtain sufficient cell-mass [5]. The cells were harvested anaerobically, washed and resuspended in an anaerobically prepared 50 mM Tris-HCl buffer, pH 8.5, containing 5 mM dithiothreitol (DTT), 0.1 mM ZnCl,, DNase (5 pg/mi) and used immediately for the preparation of crude cell extract as described earlier [5]. The protein was determined by the Biuret method [6] or Lowry’s method 171. 2.3. Enzyme assay The activity of ALA dehydratase was determined by measuring the product porphobilinogen formed under nitrogen atmosphere [S]. One unit of enzyme activity is defined as the formation of 1 pmol of PBG per hour. The effect of various metal ions, thiol compounds, was studied by incorporating them into the assay mixture at appropriate concentrations. The metal ion-depleted enzyme was prepared as described by Borralho et al. [8]. 2.4. Purification of ALA dehydratase The purification of ALA dehydratase from M. barkeri was performed under N, atmosphere at 4”C

Table 1 Purification

of 5-aminolevulinic

Purification

step

Crude enzyme Protamine sulphate Ammonium sulphate Sephadex G-25 DEAE sephacel

acid dehydratase

from 5 g of wet cells. A cell-free extract was prepared as described, and protamine sulphate solution (15 mg/ml) was added slowly to reach the ratio of 10% (v/v). After 10 min, any resulting precipitate was removed by centrifugation at 38 000 X g. The supernatant was fractionated by the addition of ammonium sulphate. The fraction that precipitated between 50 and 55% saturation contained most of the enzyme activity. The enzyme was collected by centrifugation at 38 000 X g for 30 min. The resultiilg enzyme preparation was found to be b-dole for at least three months when kept as a paste at 0” C under a nitrogen atmosphere. The precipitated enzyme was dissolved in 50 mM Tris-HCl buffer, pH 8.5, containing 50 mM DTT and 0.1 mM ZnCl, and desalted on a Sephadex G-25 column. Desalted enzyme solution was further fractionated on DEAE-Sephacel column (1.5 x 8.0 cm>, previously equilibrated with the same buffer. The proteins were eluted from the column in ten steps with the above buffer containing 0.1 M to 1.0 M NaCl. Active fractions were pooled and precipitated with ammonium sulphate at 70% saturation and the resulting precipitate was collected by centrifugation. The precipitated enzyme was found to be stable at 4”C in an atmosphere of N,. The enzyme precipitate from this step was dissolved in the initial buffer, desalted and used for subsequent studies. 2.5. Polyacrylamide

gel electrophoresis

Disc gel electrophoresis of native proteins was carried out with 7.5% acrylamide in 0.1 M Tris/HCl buffer (pH 8.3). The gels were run with 2 mA current per tube at 4”C. Protein bands were stained with 0.25% Coomassie blue [9]. Activity staining of

of Methanosarcim barkeri

Total volume (ml)

Total units pmol PBG h-’

Total protein (mg)

Specific activity (units/mn protein)

Yield (%)

10.5 9.0 5.0 6.0 5.0

210 240 225 162 70

44 30 21 13 3

4.8 8.0 10.7 12.5 23.3

100 100 93 67 29

Wet weight of cells 5.0 g. Cells were sonicated

for 3 min in each cycle.

S. Bhosale et al. / FEMS Microbiology Letters 127 (1995) 151-155

the gels was performed according to Gibbs et al. [2]. SDS gel electrophoresis was performed according to Laemmli [lo].

3. Results and discussion ALA dehydratase from cell-free extract of M. was purified to apparent homogeneity as revealed by polyacrylamide gel electrophoresis. Results of a representative purification of ALA dehydratase from 5 g of wet cells of M. barkeri are summarized in Table 1. Protamine sulphate treatment caused an apparent 14% increase in total units of the enzyme, indicating removal of an inhibitor of the enzyme by this step. Similar observations have also been reported in the case of yeast enzymes [B]. The total activity after protamine sulphate treatment was considered as 100%. During ammonium sulphate fractionation, fraction precipitating of between 5055% saturation and containing most of the enzyme activity, was desalted. Fractionation of desalted enzyme on DEAF-Sephacel column resulted in eluting

barkeri

153

ALA dehydratase as a single peak at 0.5 mM NaCl concentration of the gradient. This enzyme preparation contained a single polypeptide species as seen by PAGE (Fig. la). Specific activity after the final step of purification was found to be 23.3 pmol h-’ (mg protein)-’ at 37” C. This is very close to that of the most highly purified mammalian enzymes (24.0 pmol h-’ (mg protein)-‘: [2,11]). However, it is very low when compared to bacterial enzymes (e.g. 97 pmol hh’ (mg protein)-’ for Rhodopseudomonas capsulata: [12]; 121 pmol h-’ (mg protein)-’ for Rhodopseudomonas spheroides: [13]; and 33.0 pmol h-’ (mg protein)-’ for recombinant E. coli: [14]). The specific activity of the isolated enzyme is higher than the enzyme from yeast which was reported to be 16.2 pmol h-’ (mg protein)-’

kJ1. The iI4, of the native enzyme by gel filtration was found to be 280000 (data not shown) while SDSPAGE of the isolated enzyme showed a single band of IV, 30000 + 2000 (data not shown). This suggests that the enzyme is composed of about eight apparently identical subunits. These values are simi-

80 70 60

Fig. 1. (a) Polyacrylamide gel electrophoresis of isolated M. barkeri ALA dehydratase. (b) The effect of pH on the activity of M. barkeri ALA dehydratase. The enzyme was assayed at various pH values in 0.2 M glycine buffer containing 0.1 mM ZnCl,, 1 mM dithiothreitol and 5 mM ALA.

S. Bhosale et al./FEMS

154

MicrobiologyLetters 127 (1995) 151-155

lar to that obtained for the human erythrocyte ALA dehydratase which has eight apparantly identical subunits of 31000 and 35000 [2,11]. Most of the ALA dehydratases from plant and bacterial sources are hexamer, consisting of six identical subunits of 50 000 and 40 000 respectively [15]. However, Spencer and Jordan [14] have reported ALA dehydratase of recombinant E. coli to be a homooctamer, of subunits of 36554 + 17 Da with a total M, of 270 000 t_ 20 000. The isolated enzyme exhibits very interesting characteristics regarding optimum pH; it exhibited two pH optima at 8.5 and 9.4 (Fig. lb). Bacterial and yeast enzymes have alkaline pH optima, 8.0 to 8.5 and 9.8 respectively [14]; however it differs from mammalian enzymes which exhibits pH optima between 6.3 and 7.1 [2,11]. ALA dehydratase from Rhodopseudomonas capsulata had an optimum pH activity at about 8-8.6. However, the activity in phosphate buffer was lower and showed two pH regions with maximal activity [12]. Bonis et al. [16] have also reported two optimum pH in a range of 7.2-7.6 and 9-10 for the ALA dehydratase from Rhizobium japonicum and Rhizobium meliloti. The isolated M. barkeri ALA dehydratase resembles mammalian enyzmes isolated from beef liver [17,18], human erythrocytes [2,11] and yeast enzyme [8] in its activation by Zn2+, and was optimally stimulated by Zn2+ ions at 0.1 mM concentration. However, higher concentrations were inhibitory to the enzyme (data not shown). Various concentrations of Co2+, Fe2+, Mg2+ and Mn2+ had no effect on the activity of the enzyme. The M. barkeri enzyme differs from plant enzymes, which require Mg2+ and Mn2+ for their activation [15]. The isolated enzyme was activated by K+ upto 3 mM concentration similar to bacterial enzymes [13]. ALA dehydratase from Rhodopseudomonas capsulata does not require metal ion for its activation [12]. Our enzyme also gets Table 2 ALA dehydratase

activity in cell extracts of M. barkeri grown on various substrates

Source of carbon and energy

Trimethylamine Methanol Acetate

activated with 0.15 mM Ni2+ which is a component of factor F4a,, present mainly in methanogens. However, the enzyme was not activated with Co2+, a component of corrinoids, present in methanogens. It was markedly inhibited by Cu2+ and Pb2+ with 50% inhibition at 3 PM and 4.5 PM concentration respectively (data not shown). These divalent heavy metal ions are known for their ability to combine with cysteinyl residues in the enzyme [2,8]. This indicated that M. barkeri ALA dehydratase contained cysteine thiol groups. M. barkeri AU dehydratase is a thiol enzyme like all other ALA dehydratases, and is maintained in an active state by reduced thiol compounds such as D-mercaptoethanol, dithiothreitol, glutathione or Lcysteine and it loses its activity unless protected by one of them. Glutathione was found effective in restoring ALA dehydratase activity followed by D’IT, B-mercaptoethanol and L-cysteine (Fig. 2). In order to examine if ALA dehydratase activity levels vary with energy and carbon source, acetate-, methanoland trimethyl amine-grown cells were assayed. Table 2 shows that the ALA dehydratase activity was much higher in acetate-grown than in methanol-grown cells. Trimethyl amine-grown cells showed the lowest ALA dehydratase level. Interestingly, it appeared that the activity of the enzyme at pH 9.4 was higher than that at pH 8.5 in cell-free extracts of acetate-grown cells. The enzyme showed an increasing trend of activity at pH 9.4 as the carbon source changes from trimethylamine, methanol and acetate respectively. Thus it appeared that the ALA dehydratase levels are regulated in relation to growth substrates. Our study demonstrates the resemblance of ALA dehydratase of M. barkeri with yeast and mammalian enzyme in relation with Zn2+ activation, molecular weight, subunits, etc. On the other hand it resembles bacterial enzymes in some respects such as pH optima and activation by K+ ions. M. barkeri

Concentration

of substrate (mM)

50 50 50

Wet weight of cells in each case is about 1.6-1.7

g. Cells sonicated

ALA dehydratase

specific activity

pH 8.5

pH 9.4

3.5 4.4 9.1

2.9 4.1 10.1

for 10 min in each cycle.

S. Bhosale et al. / FEMS Microbiology Letters 127 (1995) 151-155 tion and properties

ix 60* C. ‘z 80c3 g 70: 60. E

21 u

5

10

20

lhiol compound(mM) Fig. 2. The effect of thiol compounds on the activity of M. barkeri ALA dehydratase. The enzyme assays were carried out in presence of various concentrations of glutathione, dithiothreitol, 8-mercaptoethanol and cysteine.

consumed acetate, methanol and trimethylamine as energy and carbon source. The ALA dehydratase could be constitutive irrespective of the substrate, though regulation and control of ALA dehydratase activity could be different on various growth substrates. The observed high levels of this enzymes in crude extracts could be related to a high corrinoid content in cells of M. barkeri as ALA dehydratase is a key enzyme in biosynthesis of corrinoids. The results reported here indicate the unique characteristics of ALA dehydratase of M. barkeri which is a member of archaebacteria. Genetically expressed proteins in archaebacteria are probably more closely related to their eucaryotic than eubacterial correspondants, as observed by others [19,20].

References HI DiMarco, A.A., Bobik, T.A. and Wolfe, R.S. (1990) Unusual coenzymes of methanogenesis. Ann. Rev. Biochem. 59, 355-394. El Gibbs, P.N.B., Chaudhry, A.G. and Jordan, P.M. (1985) Purification and properties of 5-aminolevulinate dehydratase from human erythrocytes. Biochem. J. 230, 25-34. [31 Gibson, K., Neuberger, A. and Scott, J. (1955) The purifica-

of Gaminolevulinic

155

acid dehydratase.

Biochem. J. 61, 618-629. 141 Gilles, H. and Thauer, R.K. (1983) Uroporphirinogen III, an intermediate in the biosynthesis of the nickel containing factor F4s0 in Methanobacterium thermoautotrophicum. Eur. J. Biochem. 135, 109-112. 151 Bhosale, S.B., Nilegaonkar, S.S., Yeole, T.Y. and Kshirsagar, DC. (1989) Evidence for existance of multiple forms of hydrogenase in Methanosarcina. Biochem. Int. 19, 10951108. 161Clark, J.M. and Switzer, R.L. (1977) Amino acids, proteins and enzymology. In: Experimental Biochemistry, 2nd Edn., W.H. Freeman and Company, pp. 67-85. [71 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. bl Borralho, L.M., Ortiz, C.H.D., Panek, A.D. and Mattoon, J.R. (1990) Purification of Gaminolevulinate dehydratase from genetically engineered yeast. Yeast 6, 319-330. 191 Clark, J.M. and Switzer, R.L. (1977) In: Experimental Biochemistry 2nd Edn., W.H. Freeman and Company, pp. 300301. [lOI Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophag T4. Nature 227, 680-685. [ll] Anderson, P.M. and Desnick, R.J. (1979) Purification and properties of S-ALA dehydratase from human erythrocytes. J. Biol. Chem. 254, 6924-6930. [12] Nandi, D.L. and Shemin, D. (19731 GAminolevulinic acid dehydratase of Rhodopseudomonas capsulata. Arch. Biochem. Biophys. 158, 305-311. 131 Nandi, D.L., Cohen, K.F.B. and Shemin, D. (1968) 6 Aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. I. Isolation and properties. J. Biol. Chem. 243, 1224-1230. 141 Spencer, P. and Jordan, P.M. (1993) Purification and characterization of 5-aminolevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal binding domain. Biochem. J. 290, 279-287. 151 Boese, Q.F., Spano, A.J., Li, J. and Timko, M.P. (1991) Aminolevulinic acid dehydratase in pea (Pisum satiuum L). Identification of an unusual metal binding domain in the plant enzyme. J. Biol. Chem. 266, 17060-17066. 161 De Bonis, A.F., Rossetti, M.V. and Battle, A.M. (19921 1’ Delta-aminolevulinate dehydratase from free living Rhizobium. Int. J. B&hem. 24, 1841-1847. [17] Bevan, D.R., Bodlaender, P. and Shemin, D. (1980) Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity. J. Biol. Chem. 255, 2030-2035. [18] Cheh, A. and Neilands, J.B. (1973) Zinc, an essential metal ion for beef liver Saminolevulinate dehydratase. Biochem. Biophys. Res. Commun. 55, 1060-1063. [19] Iwabe, N., Kuma, K.I., Hasegawa, M., Osawa, S. and Miyata, T. (1989) Evolutionary relationship of archaebacteria, eubacteria and eucaryotes inferred from phylogenotic trees of duplicated genes. Proc. Natl. Acad. Sci. USA 86, 9355-9359. [20] Zillig, W., Palm, P., Reiter, W.D., Gropp, F., Puhler, G. and Klenk, H.P. (1988) Comparative evolution of gene expression in archaebacteria. Eur. J. Biochem. 173, 473-482.