JOURNAL OF BACTERIOLOGY, Junie 1967, p. 1770-1776 American Society for Microbiology

Vol. 93, No. 6 Printed ii U.S.A.

Copyright ( 1967

Fumarate Reductase Activitv of

Streptococcus faecalis B. J. AUE AND R. H. DEIBEL' Divisiont of Biologicacl Scienices aitd Departnielit of Food Sciettce, College of AIgricultu/ure, Corltc/ll Ititoca, New Yor-k 14850

Ltlierasil,

Received for publication 13 JanuLary 1967

Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The K,0 value of the enzyme for reduced flavin mononucleotide was 2 X 10-4 as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. Ni

The reduction of fumarate by various obligate and facultative anaerobes has been described and the responsible enzymes have been purified and characterized from yeast (T. P. Singer and J. Hauber, Federation Proc. 24:297, 1965), Micrococcus lactilyticus (14, 15), and Escherichia coli (7; C. A. Hirsch, Federation Proc. 24:228, 1965). These enzymes, with the exception of Enzymes II and III from yeast, are capable of catalyzing the oxidation of succinate as well as the reduction of fumarate. Although Streptococcus faecalis does not possess iron-porphyrin enzymes or a functional tricarboxylic acid cycle, an active fumarate reductase has been reported (6). Evidence for the oxidation of reduced nicotinamide adenine dinucleotide (NADH2) by fumarate in this organism, which probably occurs via a flavin intermediate, was observed subsequently (8). When S. faecalis is grown with substrate quantities of fumarate, a radical alteration of the normal homofermentative end products is effected (3). The end products of this diverted fermentation suggest the reduction of fumarate to succinate and an altered metabolism of pyruvate to yield volatile acids and carbon dioxide instead of lactate. Under these conditions, it was assumed that fumarate, rather than pyruvate, was associated with the regeneration of oxidized nicotinamide I Present address: Department of Bacteriology, The University of Wisconsin, Madison.

adenine dinucleotide. The inability of S. Jaecalis to degrade the carbon skeleton of fumarate and the absence of a fumarase in this organism was reported previously (2). Some sensitivity of the fumarate reductase to acidity was observed with cell suspensions. However, common metabolic inhibitors such as azide, cyanide, arsenite, atabrin, and malonate did not effect an inhibition of activity (3). At the cellular level, no significant oxidation of succinate was demonstrable, and definitive evidence regarding the inducible or constitutive nature of the enzyme was not obtained. The current investigation was undertaken to extend and reinforce previous studies with regard to the fumarate reductase from S. faecalis. MATERIALS AND METHODS

Straici. S. faecalis FB82 was used througlhout this

study. The source and characteristics of this strain have been described previously (4). Metlia atid cultire coliditionis. Cells for the cell-free extracts were grown under stationary conditions for 8 hr at 37 C in a broth containing 1%,"/ Tryptone (Difco), 0.5%," yeast extract (Difco), 0.5%,, sodium chloride, 0.5%- K2HPO4, and 0.2%' glucose (pH 8.0). Cells grown in this broth were lyrophilized and stored (- 15 C) over a desiccant. For stuLdies regarding the inducible nature of the enzyme, a semisynthetic medium (5) was employed with andcc without added fumarate (0.5 ). Antalytical ntet/tods. IProtein was estimated by the trichloroacetic acid lprecipitation method (I 3), and by 1770

absorption measurement of the solution at 260 and

Beckman DU-2 spectrophotometer. Fumarate reductase activity was measured at 470 m,u by the method of Jacobs and Vandemark (8). Specific activity is expressed in micromoles of reduced flavin mononucleotide (FMNH2) oxidized per min per mg of protein. The phenazine methosulfate assay (1) was employed to study the oxidation of succinate to fumarate. All assays were performed under helium. Prepalrationl of cell-free extracts. Crude extracts were prepared by suspending 0.8%7C (dry weight) of cells in a solution containing 0.1 M potassium phosphate and 0.01 NI sodium thioglycollate (pH 7.4). For purification procedures, 1.6'%0 (dry weight) of cells was used. The cells were disrupted by treatment in a 10-kc Raytheon sonic oscillator for 15 to 20 min in volumes of not more than 50 ml. Cellular debris was removed by centrifugation at 31,000 X g for 1 hr at 2 C. Elizvme putrificationt. A 6% protamine sulfate solution (pH 6.7) was heated to 50 C, and 0.27 ml/100 mg of protein was added with stirring to the crude extract (2 C) to precipitate nucleic acids. To each 100 ml of crude extract at 2 C, 18.9 g of solid ammonium sulfate was added slowly with stirring to yield a 35%" saturated solution. The precipitate was removed by centrifugation and discarded. To the remaining solution, 6.23 g of additional ammonium sulfate was added, yielding a 45% saturated solution. This precipitate was removed by centrifugation, suspended in 0.01 sodium thioglycollate, and dialyzed against a solution containing 0.01 N4 potassium phosphate (pH 7.4) and 0.01 Ni sodium thioglycollate at 2 C for 2 hr. The 35 to 45' ammonium sulfate fraction contained most of the fumarate reductase activity. The ammonium sulfate fraction (35 to 45% 0 saturation) was placed on a column1 of Bio-Gel (Calbiochem, Los Angeles, Calif.) consisting of 2.5 by 22 cm of P-300 under a layer of 2.5 by 3 cm of P-60. A buffer (pH 7.4) containing 0.01 Ni lpotassium phosphate and 0.01 N/I sodium thioglycollate was used as the eluting

280 m,u with

1771

FUMARATE REDUCTASE OF S. FAECALIS

VOL. 93, 1967 a

NI

an initial pH of 8.0 (prior to autoclaving), coupled with an 8-hr incubation period. Under these conditions, a specific activity of approximately 0.14 was obtained. Preservation of the enzyme for study. The fumarate reductase activity was stable to lyophilization of the whole cells, and these preparations were used as the source of the enzyme. Prolonged contact of the cell-free preparation with air resulted in a marked loss of activity. This difficulty was circumvented by the addition of 0.01 M sodium thioglycollate to the cell-free extracts. Long-term storage was accomplished by freezing the cell-free extracts and thawing them at refrigerator temperature for use. The frozen extracts displayed approximately a 10%C loss in activity after 2 weeks of storage, as compared to a 53 % loss in refrigerated extracts. Various reducing agents including reduced glutathione, dithiotreitol, and sodium thioglycollate were tested for their ability to enhance fumarate reductase activity (Table 1). The most effective reducing agent was thioglycollate at a concentration of 0.01 M, and it was employed in the majority of subsequent experiments. Crude cell-free extracts prepared with 0.01 M thioglycollate resulted in specific activities usually ranging from 018 to 0.28. Effect of pH and ionic strength. A peak in activity was observed at pH 7.4, although there was measurable activity at pH 5.0 and 8.0 (Fig.

reduicinig agen-ts otn fiumarate reduictase activity dlutrinig preparcationl of

TABLE 1. Effect of

cell free extractsa Reducing agent

agent.

RESULTS

Optimal growth conditions for fiumarate reductase production. An acidic pH value of the culture medium was shown previously to have a detrimental effect on the fumarate reductase activity of the cell crop (3). To obtain maximal specific activity, various growth media were prepared by use of the Tryptone-yeast extract-phosphate basal medium (Materials and Methods). The glucose and fumarate concentrations, as well as the initial pH value of the medium and the incubation period, were tested for their effect on the specific activity of the cell-free preparations. Fumarate had no significant effect on the specific activity, and in subsequent experiments it was omitted from the growth medium. In general, low final pH values (below 5) adversely affected activity. The most active preparations were obtained when the growth medium contained 0.2% glucose with

RedLiced glutathione Dithiothreitol

Concn

(mm)

10

10 5 1 0.5

Thioglycolate

None

20 10 5 1 -

Specificb

atvt

0.17 0.18 0.12 0.18 0.14

0.18 0.23 0.18 0.16 0.16

a Cuvettes contained 0.6 ymoles of FMNH2, 20 ,umoles of sodium fumarate, 120 /moles of phosphate buffer (pH 7.4), 1 mg of protein (crude extract containing 0.01 NM sodium thioglycollate), and distilled water to 3.0 ml. Reducing agents were added to the cell suspensions before sonic

treatment.

b Specific activity = micromoles of FMNH2 oxidized per min per mg of protein.

1772

AUE AND DEIBEL

J BAc-rERIOL .

025

e 0.20 0.15-

5:

0.15 u0.10-

I uJ

Q-

00

005

4.0

(1)

5.0

6.0

pH

7.0

8.0

9.0

FIG. 1. Effect of pH on fiumarate r-edlluctase activity. Cuvettes coiitainled 120 j,moles of the aippropriate buffer, 0.6 ,moles of FMNH2, 20 p,moles of fiamarate, and I mg of protein (crude extract contaitilnlig 0.01 M thioglycollate), with distilled water to 3 nil. The followintg buffers were used: acetate (pH 4 anid 5), phosphiate (pH 6 to 7.8), anid tris(hydroxyml1etlhyl)amlitnom7iethbane (pH 8 and 9).

1). Activity decreased more rapidly with basic than with acidic conditions of assay. Ionic strength variation of the phosphate buffer from 0.073 to 0.095 (100 to 130 ,umoles per cuvette) revealed an optimum approximating 0.088 (Fig. 2). For these determinations, the enzyme was incubated with the appropriate buffer for 10 min before the reaction was initiated by the addition of fumarate. Inducible nature offumarate reductase. Growth in the semisynthetic, acid-hydrolyzed casein medium with and without added fumarate confirmed early results with complex media that there was no significant increase in fumarate reductase activity as a result of growth with fumarate (Table 2). These results indicate a constitutive nature for the enzyme. Substrate specificity. In addition to fumarate, maleate and mesaconate were utilized as substrates (Table 3). The following could not be hydrogenated: crotonic, cinnamic, trans-, transmuconic, trans-3-hydromuconic, ferulic, tiglic, angelic, itaconic, citraconic, and acrylic acids. Inhibitors. A number of carboxylic acids were tested for inhibitor activity. Succinate, malonate, citraconate, and trans-, trans-muconate inhibited the reduction of fumarate, whereas crotonic, cinnamic, acrylic, aspartic, trans-4-hydromuconic, ferulic, and itaconic acids did not inhibit (Table 4). No inhibition was observed with these compounds unless the inhibitor was incubated with

0.051

0073 0083 0.088 0095 IONIC STRENGTH FIG. 2. Effect of ioniic strenigth ont fiut,nrate reducta.se activity. Cuvettes contained 100, 110, 120, or 130 u.Lnoles of phosphate buiffer (pH 7.4), 0.6 pinoles of FMINH2, 20 j.unoles of fium1arate, 1 ing ojf proteini (35 to 45%,, saturated anunoniunm sulfate fractionz in 0.01 AI

thioglycollate) an1d dlistilled water to 3

nml.

TABLE 2. Effect of growth with filumarate on7 fiu??arate redluctase activity, Growth medium

Specific activitvy'

Semnisynthetic ................ Semisynthetic + 0.5%-

0.268. 0.270,

fumarate....

0.263, 0.266c

See footnote a, Table 1. See footnote b, Table 1. Duplicate preparations.

both the FMNH2 and the enzyme preparation prior to the addition of fumarate. Other inhibitory agents including cyanide, azide, ethylenediamine tetraacetic acid, and 2,2'bipyridine at a concentration of 5 X 10'!I did not inhibit enzyme activity. lodoacetate ,-t I X 10-3 M and p chloromercuribenzoate at 1 10-' M effected a limited inhibition (Table 4). Activation by amino acids. Certain amino acids, when added to the reaction mixture in the amount of 10 ,moles, produced an increase in the specific activity of the extract. Glycine, alanine, histidine, and possibly lysine were stimulatory (Table 5). Serine, aspartic acid, and arginine were not. Growth with 0.1% glycine in the medium did not increase the fumarate reductase activity of the extracts. Purification. Purification was accomplished by treating the crude extracts with protamine sulfate and then fractionating with ammonium sulfate. The 35 to 45% saturated fraction was dialyzed and passed through a Bio-Gel P-300 column as x

FUMARATE REDUCTASE OF S. FAECALIS

VOL. 93, 1967

TABLE 3. SecondarY, suibstr-ates Jor fimiarate r edulctase Sub)stratc

Fumarate ....... Maleate ......... Fumarate ....... Mesaconate...

E' XI tSpcific activityb

7OxIno 1

l 2 2

0.148 0.059 0.166 0.084

TABLE 4. Effect

of

i/ihiitors onz fiumarate activity

Per cent of

bv fumarate

100 40 100 51

nXIt

Inlhibitor

reaction given

1 773

Concn (11)

Nonea

Tral;is-,

0.224e

-

Succinate Malonate Citraconate

6.7 X 1041 0.191 6.7 X 10 0.183 6.7 X 10- 0.171 -

6.7 X

trails-

muconate

Noneb

--

2,2'-Bipyridine Sodium cyanide Ethylenediamine-

5 X 5 X

Noneb' c p-Chloromercuri-

0.151

0

10-