Delta-aminolevulinate dehydratase from free-living Rhizobium

0020-71 IX/92 S5.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd

fnt. J. Biochem. Vol. 24, No. 11, pp. 1841-1847, 1992

Printed in Great Britain. All rights reserved

DELTA-A~INOL~V~LINATE DE~D~TASE FREE-LIVING RHIZOBIUM

FROM

A. F. DE BONIS, M. V. ROSSE~ and A. M. DEL C. BATLLE* Centro de lnvestigaciones sobre Porfirinas y Porfirias-CIPYP, (CONICET, FCE y N, UBA), Ciudad Universit~a, Pabellitn II, 2 do Piso, 1428 Buenos Aires, Argentina (Received 29 October 1991) Abstract-l. 6-Aminolevulinate-dehydratase (ALA-D) from Rhizobiumjaponicum and Rhizobium meliloti was isolated and some properties were studied. 2. The enzyme from both strains require DTT to maintain full activity and a concentration of about 8 mM is necessary for its maximum expression. Thiol inactivating compounds and heavy metals ions such as Pb*+ and Cd2+ inactivate ALA-D. 3. The enzyme exhibits Michaelis-Menten kinetics and has an apparent K, of 0.095 and 0.1-0.37 mM for Rhizobium japonicum and Rhizobium meliloti respectively. 4. For both strains the pH profiles show a well defined maximum at about 7.2-7.6 and a second broad peak or shoulder in the range of pH 9-10. 5. ALA-D from ~izobium does not appear to be a heat stable enzyme as it happens to be in other

sources

I~RODU~ON

Bacteria of the genera Rhizobium form nitrogenfixing associations with leguminous plants. Symbiosis begins with the recognition and invasion of the roots of the legume host by Rhizobium followed by proliferation and differentiation of both organisms to develop highly specialized structures known as root nodules. It is within these root nodules that conditions for bacterial nitrogen fixation and for host plant nitrogen assimilation are established. Effective nitrogen fixing symbiosis involving Rhizobium requires the presence of Leghemoglobin (Lb) (Appleby, 1984). This hemeprotein is only found in nodules, neither partner is able to produce Lb when grown separately. The apoprotein of Lb is encoded in the plant genoma as indicated by both chemical synthesis and analysis of isolated genes. The heme moiety is thought to be synthesized by the bacteria (Cutting and Schulman, 1969; Godfrey and Dilworth, 1971). However, the tetrapyrrole biosynthetic pathway which leads to the formation of corrins, cytochromes, bilins and chlorophyls as well as heme, is also operational in plants, and the possibility that heme precursors are transferred between the symbiotic partners has not yet been excluded (~ue~not and Chelm, 1986; O’Brian et al., 1987). Data about Lb biosynthesis in nodules are rather controversial. In

some cases it has been reported that bacteria are more active than plant in heme synthesis, while in others heme forming capacity appears to be equally effective in either component (Nadler and Avissar, 1977; Leong et al., 1982; Guerinot and Chelm, 1986). Therefore, the study of heme biosynthesis in Rhizobium seems to be an attractive area for research. It is also wo~hwhile emphasizing that, although a great deal of information is available on this pathway in most sources, very little is known, however, in Rhizobium (Keithly and Nadler, 1983; Guerinot and Chelm, 1986; Jacobs et al., 1989). Therefore, with the aim of elucidating the true roles that the symbiotic partners play in Lb-heme moiety synthesis, we have initiated a study of the enzymes involved in its biosynthesis in both, free-living and symbiotic bacteria. We describe here some properties of the second specific enzyme of this pathway, &aminolevulinic acid-dehydratase (ALA-D), which catalyzes the condensation of 2 molecules of ALA to the heterocyclic monopyrrole porphobilinogen (PBG), in 2 strains of free-living Rhizobium which differ from each other in their growth properties.

MATERLALSAND METHODS

*Address correspondence to: Professor Dr Aicira Batlle, Viamonte 1881 10” “A”, 1056 Buenos Aires, Argentina.

Tris-HCl and phosphate buffers were used throughout this study, unless otherwise indicated. ALA was from Sigma Chemical Co., U.S.A. Other reagents employed were of the highest purity commercially available. Solutions were made up in ion-free, 3 times distilled water.

1841

A. F.

1842

DE BONIS et al.

in the resulting supematant PBG was estimated according to Moore and Labbe (1964). Protein concentration was determined by Bradford’s method (Bradford, 1976). One enzyme unit is defined as the amount of enzyme that catalyzes the formation of 1 nmol PBG/hr from ALA under the standard incubation conditions. Specific activity is expressed as the number of units of enzyme/mg of protein. RESULTS

Optimal assay conditions for ALA-D

A well defined maximum of enzyme activity in early exponential and stationary phase of growth was observed for Rhizobium japonicum and Rhizobium met’iloti respectively (Fig. 1).

60

60

40

20

Time (hrl

Fig. 1. Effect of growth on enzyme activity. At indicated times aliquots from R. japonicum (0, +) and R. meliloti (*, 0) cultures were removed for optical density (O.D.) (0, Jle) and activity (+, 0) measurements. All methods employed were those described in Materials and Methods. Preparations of cell-free extracts Free-living cells of Bradyrhizobium japonicum 110 USDA (low growth) and Rhizobium meliloti 102F (fast growth) were grown, unless otherwise indicated, at 30°C in YEM broth (1% manitol; 0.2% yeast extract; 0.05% K,HPG,; 0.02% MgSO,.7H,O; 0.01% NaCl) for about 72 hr to late log phase for Bradyrhizobiurn japonicurn and 48 hr for Rhizobium meliloti. Cells were harvested by centrifugation at 10,OOOgfor 10 min, washed once with 30 mM Tris-HCI buffer pH 7.8 and suspended in the same buffer containing 10 mM DTT (1: 3 wet wt/v). Cells were then disrupted by ultrasonic treatment (3 min, 10 p A; MSE sonicator) and the homogenate (H) obtained was centrifuged at 15,OOOgfor 10 min. The pellet (P) was discarded and the supematant (S) was employed as enzyme preparation.

ALA-D activity was almost completely associated with the supernatant fraction. It was also found that specific activity in the supematant was the same in both anaerobic (R. japonicum: 1.82 U/mg; R. meliloti: 2.05 U/mg) and aerobic (R. japonicum: 1.87 U/mg and R. meliloti: 2.15 U/mg) incubation atmosphere. It was observed early that a thiol protective reagent such as DTT had to be added to the extraction buffer to obtain an active enzyme preparation. Moreover, as can be seen in Table 1, the protective effect of DTT was higher when the enzyme was preincubated with this thiol; its optimum concentration was 8 mM. However, unexpectedly GSH and cysteine inhibited the enzyme at all concentrations tested. ALA-D activity of this crude enzyme fraction was very stable, it remained practically unchanged after being kept for 7 days and retained 3040% of the initial values after 1 month storage. In both strains, PBG formation was linear with protein concentration up to 2 mg and with incubation time up to 120 and 60min for R. japonicum and R. meliloti respectively.

Estimation of enzyme activity

Effect of substrate concentration-kinetics

Unless otherwise specified, the standard incubation system included: 0.45 ml of toe3 M Zn acetate; 0.5 ml of O.OSM ALA; 0.25 ml of cell-free extract; 0.25 ml of 0.2M Tris-HCl buffer pH 7.8. Incubations were carried out aerobically, in the dark, with mechanical shaking at 37°C for 1 hr. After incubation, 10% TCA was added and the inactivated protein was separated by centrifugation at 15,OOOgfor 10 rnin;

It was found that velocity vs ALA concentration plots for both strains of Rhizobium were hyperbolic at all phases of growth. Saturation was reached at about 2.5-3 mM (Fig. 2). Double reciprocal plots were linear and apparent K, values were: 0.095 and 0.1-0.37 mM for R. japonicum and R. meliloti,

Table 1. Effect of dithiothreitol (Dm Rhizobium

DTT (mM) 1 2 4 6 8 10

japonicwn

With preincubation

ALA-D activity Without preincubation

on ALA-D activitv from Rhizobium Rhizobium

meliloti

With preincubation

ALA-D activitv Without prckcubation

Units/ml

%

Units/ml

%

Units/ml

%

Units/ml

%

22.40 29.12 35.84 41.44 43.68 42.56 42.56

100 130 160 185 195 190 190

26.35 31.62 36.89 38.21 44.80 31.62 15.81

100 I20 140 145 170 120 60

10.23 21.39 31.62 58.28 66.03 73.16 70.48

100 209 309 570 645 715 689

36.27 56.42 88.66 loo.44 112.22 100.72 74.20

100 155 244 277 309 178 205

Supcrnatant from heating (5 min at 45°C for R. mdiloti; IO min at 40°C for R. japonicum) was employed as enzyme source. DTT was added at the concentrations indicated 30 min before or with the substrate. Methods used were those described in the text.

ALA-D from Rkzobium

20

1843

25

15 Jy20 v

g

10

15

s 10

I-

5 5

./

0

0

2

6

4 ALA

0

(mM)

t_ 5

Fig. 2. Effect of substrate concentration. ALA-D activity from R. juponicum (0) and R. mefifoti(e) was measured with varying amounts of ALA as indicated. All methods used were as described in the text. The inset shows Lineweaver-Burk and Hill plots. respectively. From Hill plots a slope of 1 was calculated, indicating the existence of at least one binding site of ALA (Fig. 2, inset). EfSect of pH

When the effect of pH on enzyme activity was studied it was found that pH profiles for both strains were different and also somehow unusual. For R. japonicum a maxims at about pH 7.2-7.6 and a second broad peak in the range of pH 9-10 were obtained. Activity slowly decreased at higher pH’s (Fig. 3). For R. meliloti a well defined maximum at pH 7.6 and a shoulder in the range of pH 9-10 were observed. Activity in this case decreased sharply at pH values below 7.0 and above 10 (Fig. 3). Enzyme activity for R. japo~i~ was 50% higher in Tris-HC1 buffer than in sodium phosphate or (A)

6

7

I

I

I

I

I

8

9

10

11

12

PH

Fig. 3. Effect of pH. ALA-D activity from R. juponicum (0, $, x, A) and R. mefifoti (+, 0, 0. VI were measured. Incubation system was as detailed in the text except that different 0.2 M buffers at the pH’s indicated were used. (e, i-) Sodium phosphate; (z#, 0) Tris-HC1; (x , 0) glicina-NaOH; (A, V) sodium carbonate-bicarbonate. ~ycin~~a0~ buffer and slightly higher than that in ~rbonat~bicarbonate btier. However ALA-D from R. meliloti appears to have approximately the same activity in all buffers used, at the same pH and concentration. When sodium phosphate buffer was used instead of potassium phosphate no significant changes were measured in ALA-D activity from either strain of Rhizobium. Effect of temperature

ALA-D for Rhizobium was quickly inactivated by heating, even at the shortest periods assayed, at temperatures above 40°C for R. japoni~m (Fig. 4A) and above 45°C for R. meliloti (Fig. 4B). One reason 90

(B) /:\+/+

+\

+ *-*

60

+I+-+

*

t

/ .-./*-.

/

1-x

2

4

6

8

I IO

I 12

I 14

I 16

IfJ $8

11 20

22

c)

111 2

4

6

I!: 8

10

1

I

1

i

!:

1

12

14

16

16

20

22

Time fmin f Fig. 4. Effect of heat treatment. Aliquots of R. juponicum(A) and R. mefifoti(B) supematants were heated at the temperatures and for the periods indicated. The activity of the control, without any treatment, was taken as 100%. Experimental conditions were as indicated in the text. (@) 37°C; (+) 40°C; (*) 45°C (13) 50°C; (x) WC.

A. F. DE BONGef al.

I844

Table 2. Effect of EDTA on ALA-D activity Activity (%)

EDTA (nW

200

Dialyzed

0

25 50 100 150 200

150

72 2 Y

s

Not dialyzed

loo

ioo

113.6 123.4 100 55 6.5

124.86 108.67 115.03 100 100

Supematant from 24,000g centrifugation was divided into 2 parts, one of them was dialyzed overnight against buffer + DTT, and in both activity was measured in the presence of increasing amounts of EDTA. The activity of each fraction incubated without EDTA was taken as 100%.

100

50

Temperature (*Cl

Fig. 5. Effect of incubation temperature. ALA-D activity from R. japonicwn (0) and R. meliloti (0) was measured at the temperatures indicated. Experimental conditions were those described in the text. In the inset Arrhenius plots are shown.

When the dialyzed supernatant (overnight, against buffer) was incubated in the absence or presence of 8 mM DTT and varying amounts of Zn*+, it was found that activity was completely recovered only in the presence of DTT and with a final concentration of about lo-’ M Zn2+. Metal concentrations above 10e4M, even in the presence of D’IT, drastically inhibited the enzyme (Table 3).

of inactivation

could have been DTT decomposition at high temperatures, therefore DTT was added to

Efect of thiol activity

incubation buffer for activity measurement. It was found that activity was the same except at the lowest temperatures assayed for R. meliloti and at 40°C for R. ja~~nicum, in either case a slight activation was observed. Only the results obtained in the presence of D’IT are shown (Figs 4A and B). When the effect of the incubation temperature on activity was studied it was found that its maximum was measured between 37-40 and SC+O”C for R. japon~cum and R. meli~oti, respectively. In the latter, a protective effect of substrate was also observed (Fig. 5). Arrhenius plots (Fig. 5, inset) indicated an important protein denaturalization at temperatures above 40-50°C for the enzyme for both strains. The same results were obtained when heat treatment was carried out in the presence of PMSF, suggesting that heat sensitivity is not due to a protease stimulation. From these plots an activation energy of 8.2 cal/mol (Q,,: 1.3) and 11.27 cal/mol (Q,,: 1.91) for R. japonicum and R. meliloti respect-

ALA-D from Rhizobium requires DTT and therefore the presence of reduced sulphydryl groups for full activity. On these grounds, it is expected that thiol inactivating compounds might have a pronounced inhibitory effect on activity (Table 4). However, when alkylating reagents (Group I) were added together with the substrate, they did not show any effect on ALA-D from Rhizobium meliloti, but inhibit the enzyme from Rhizobium japonicum by about 50-~%. When the enzyme was preincubat~ with the reagent, in the absence of ALA, the effect was generally more pronounced especially in

ively was calculated, sensitivity to heating.

indicating

again

the

high

Effect of chelating agents and zinc ions

Activity was measured in the supernatant before and after a dialysis treatment (overnight, against buffer $ DTT), in the presence of increasing amounts of EDTA. It was found that only the dialyzed supematant was inhibited by EDTA at concentrations above 150 mM. These results suggested that the enzyme, which is a known metallo-enzyme, would be well protected to the action of chelating agents in this crude preparation. After dialysis ALA-D became more sensitive to its action (Table 2).

inactivating

chemicals

on ALA-D

R. meliloti.

As for the oxidative reagents (Group II), the action of DTNB was again greater on R. japonicum in either condition, while the enzyme from R. ~liloti was

Table 3. Effect of zinc and DTT on ALA-D activity [Zn’+] (nw 0 10-s

2.5 x 5.0 x 10-4 1.5 x 2.0 x 2.5 x

10-s 10-S lo-’ 10-4 10-h

Activity (%) +DTT

-DTT

0

87

20 20 19 10 5 5 4

100 112 109 97 81 26 8

Supematant from dialysis (overnight against buffer) was used as enzyme source. Activity was measured with increasing amounts of Zn’+ as indicated (final concentrations), in the absence or presence of 8 mM DTT. The activity of supematant without dialysis was taken as ItlO%.

ALA-D from Rhizobium

1845

Table 4. Effect of sulphydryl inactivating chemicals on ALA-D activity Activity (%) Reagent ChOUV

I

Without pnincubation mM

R. iamwicum

R. meiilori

R. iooonicum

R. meliloti

PCMB

1

59.80 53.45 48.27 69.63 59.65 43.79 72.29 56.27 35.80

100.00 100.00 100.00

0 (87.50) 0 (22.50) 0 (17.47) 60.00 (0) 58.30 (0) 46.66 (0) 73.33 (0) 30.83 (0) 0 (0)

89.00(100) 90.00 (100) 85.00 (100) 44.40 (0) 39.40 (0) 36.95 (0) 46.11 (0) 30.20 (0) 0 (0)

13.42 10.22 11.68 20.00 0 0

86.26 75.53 69.26 11.62 0 0

13.23 (0) 9.16 (0) 10.64 (0) O(lOO) 0 (100) 0 (100)

48.89 (0) 25.00 (0) 0 (0) 0 (100) 0 (100) O(100)

1 5 10 0.1 1

79.56 67.17 39.56 0 66.42 0

98.16 97.15 97.13 97.30 24.46 8.26

5

0

71.66(O) 74.16 (0) 30.00 (0) 0 (0) 66.45 (90) 0 (59) tJ (35)

75.55 (0) 76.11 (0) 72.22 (0) 0 (0) 0 (28) 0 (26) 0 (25)

2.5 5

II

IA

1

NEMI

2.5 5 10 20 50

DTNB

1 2.5 5

IBZ

I

2.5 5 III

With preincubation

Addition

Pb

Cd

0.1

96.94 100.00 90.52 100.00 96.50 50.20

0

Supcmatant from heating, as indicated in the footnote to Table 1 was used as enzyme source. Reagents, in the concentration shown, were added 30min before or together with the substrate, for experiments with or without prcincubation respectively. Other conditions are those indicated in the text. Activity is expressed as a percentage of the appropriate control value, which is considered as 100%. When reversion was tested, 6 mM DTT plus ALA were added after preincubation and the percentage of reversion (indicated in parentheses) was calculated on the basis of the mpective control without Dl’T.

more inhibited when it was preincubated with the reagent, but in neither case could the effect be reverted. Iodosobenzoate almost completely inhibited the enzyme under all conditions, but its effect could be overcome. In Group III (heavy metals) 5-10 mM Pb*+ inhibited the enzyme from R. japonicum, with or without preincubation, and reversion was not observed. In R. meliioti inactivation was found only when the metal was added before the substrate, and again the effect can not be overcome. Finally, Cd*+ completely inhibited the enzyme from both strains and under all the conditions assayed; only partial reversion could be attained. Possible regulatory role for ALA-D from Rhizobium

It has been found that ALA-D activity from and R. meliloti, 19.7 and 57.0 U/mg, respectively, is as low as, or of the same order as that reported for the enzyme from organisms such as Euglena grucilis: 90 U/mg (Stella, 1977); Neurosporu crassu: OS-3 U/mg (Muthukrishman et al., 1972); Succharomyces cerevisiae: 5-19 U/mg (Barreiro, 1967; Borrahlo et al., 1983) and Mucor rouxii: 2 U/mg (yeast), 17 U/mg (mycelium) (Paveto et al., 1989). The enzyme plays a regulatory role, suggesting therefore that in Rhizobium the rate limiting step in heme pathway might be at the level of ALA-D. However, further studies on the properties and molecular structure of the Rhizobium enzyme in different strains as well as measurement of ALA-S R. japonicum

activity are necessary assumption.

to demonstrate

the above

DISCUSSION

In the first part of this study, optimum conditions for measuring PBG formation from ALA by ALA-D, were established. It has been reported that ALA-D activity might exhibit remarkable changes during organism development (Batlle et al., 1975; Labbe-Bois and Volland, 1977; Mattoon et al., 1978; Paveto et al., 1989). When studying the effect of growth on ALA-D activity from Rhizobium, different profiles for both strains were obtained. A well defined maximum at early exponential and stationary phase of growth was found for R. japonicum and R. meliloti, respectively. ALA-D from both strains of Rhizobium showed a Michaelis-Menten kinetic behaviour in agreement with the results found for the enzymes from other sources (Borralho et al., 1990). Apparent K,,, value obtained for the enzyme from R. meliloti was of the same order as those reported for ALA-D from other sources (Shibata and Gchiai, 1977; Borralho et al., 1983, 1989), but the value for the enzyme from R. japonicum was one order lower than those and similar to the value reported for Mycobacterium Phlei (Yamasaki and Moriyama, 1971). The optimum pH of ALA-D is in the range 6.3-6.8 for the mammals enzyme (Gibbs et al., 1985); 7.2-7.8 for plants enzyme (Shibata and Ochiai, 1977; Shetty and Miller, 1969); 8-9.2 for the bacterial ALA-D (Nandi et al., 1968; Yamasaki and Moriyama, 1971;

1846

A. F. DE bNI.9

Nandi and Shemin, 1973; Shemin, 1976); 8.5 for algae (Tamai et al., 1979) and 9.2-9.8 for the yeast Succharomyces cereoisiae (Barreiro, 1967; Borralho et al., 1990). These differences in optimum pH’s would indicate differences in the appropriate ionization state of the enzyme or in the enzyme-substrate complex required for activity. For the 2 strains of Rhizobium here examined pH profiles showed 2 maxima, the ratio between them being quite different. However, it is worth noting that one peak was in the region of pH 7.2-7.6 as if it corresponds to a vegetable tissue; while the other was in the range 9-10, where it is often found for microorganisms. These findings would suggest that it is very likely that, in Rhizobium there exist 2 isoenzymes of ALA-D. In all systems so far examined, ALA-D is a cytoplasmatic and heat stable protein. It is also a Zn-metallo and sulphydryl enzyme. Activity is rapidly lost on exposure to air oxidation of -SH groups or by their inhibition with classical reagents and heavy metals or by displacement of zinc by lead or by chelation with EDTA. We have observed that ALA-D from Rhizob~~ is also a citoplasmatic and sulphydrilic enzyme as indicated by the requirement of a thiol protective reagent during protein extraction and also when the effect of DTT on the dialyzed supernatant was studied. However, this thiol could not be replaced by other agents such as cysteine or GSH. The sulphyd~lic nature of the enzyme was confirmed when the action of several -SH inactivating chemicals were tested. It was found that practically all of them affected the enzyme, their effect being more drastic when they were added before the substrate. ALA-D from R. juponicum seemed to be more susceptible than the enzyme from R. meliloti to their action. Oxidative agents and Cd2+ produced the most significant effect. However, synthesis of PBG from ALA by extracts of free-living bacteria was unaffected by aerobiosis, probably due to the use of a crude preparation and or the presence of DTT in the enzyme fraction. As it was expected this crude preparation of ALA-D did not require the addition of Zn2+, but dialyzed enzyme only recovered its activity in the presence of lo-’ M Zn*+, and in the presence of DTT, indicating that like the enzymes from other sources ALA-D from Rhizobium is also a Zn-enzyme. On the other hand, ALA-D is generally a protein quite resistant to heat treatment, however, the enzyme from Rhizobium seemed to be more sensitive to the effect of heating than other ALA-D’s, considering that crude preparations were used and that a protecting effect of substrate, usually shown only in purified preparations, was observed. Moreover, the enzyme from R. me~i~otiseemed to be more resistant to heat treatment than the other, which was inactivated at lower temperatures, even in the presence of ALA. We can speculate that the differences found in some properties between the 2 strains of Rhizobium could be accounted for different velocity of growth

el

al

and perhaps, some as yet undetermined differences in heme metabolism itself. In many organisms, such as mammals (Granick and Sassa, 1971), birds (Sinclair and Granick, 1975) and certain bacteria (Burnham and Lascelles, 1963; Clark-Walker ei al., 1967), control of the biosynthesis of porphyrins and hemes involves regulation of 6 -aminolevulinate synthase (ALA-S), which catalyses the first reaction in the pathway. A regulatory role for the second enzyme in the sequence, ALA-D, has been considered unlikely because it is present in high, non-limiting amounts in such organisms. However, evidence for a regulatory role has been indicated for Propionibacterium shermanii (Menon and Shemin, 1967); Euglena gracilis (Ebbon and Tait, 1969); Neurosporu crassa (Muthukrishman et ai., 1969, 1972); ~ucch~romyce~ cerenisiue (Jayaraman, 1971; Mahier and Lin, 1974; Mattoon et al., 1978; Borralho et al., 1983, 1989) and Mucor rouxii (Paveto et al., 1989). Considering, therefore, that the activity value of this preparation of ALA-D is of the same order as similar enzyme preparations from these other sources, we expect that the enzyme from Rhizobium could also play some role in heme biosynthesis regulation. Acknowledgements-Alcira M. de1 C. Bathe and Maria V. Rossetti hold the post of Scientific Researchers in the Argentine National Research Council (CONICET). Antonio F. De Bonis was Research Fellow from UBA. This paper is part of the Doctoral Thesis of A. F. De Bonis to be submitted to the University of Buenos Aires for his Ph.D. This work was supported by grants from the CONICET. REFERENCES

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ALA-D

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