Arginine dihydrolase pathway in Lactobacillus buchneri: a review

Biochimie 70 (1988) 367-374 © Soci6t6 de Chimie biologique/Elsevier, Paris

367

Arginine dihydrolase pathway in review

Lactobacillus buchneri: a

Miguel C. MANCA DE NADRA i, Aida A. PESCE de RUIZ HOLGADO ~and Georgio OLIVER ~*

Facuitad de Bioquimica, Quimica y Farmacia, Universidad Nacional de Tucumtin, Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145, 4000 Tucumdn, Argentina (Received 24-7-1987, accepted after revision 2-11-1987)

Summary - The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO~0 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and. carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight ofthe enzymes was 199,000, 162,000 and 97,000 for ar.ginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deimmase, maximum enzymatic activity was observed at 50°C and pH 6; for ornithine transcarbamylase it was observed at 35°C and pH 8.5, and for carbamate kinase at 30°C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, AG* values were : 8,700 cal mol-~ below 50°C and 380 cal mol-! above 50°C; for ornithine transcarbamylase, the values were: 9,i00 cai mol-i below 35°C and 4,300 cal mol-~ above 35°C; for carbamate kinase, the activation energy was : 4,078 cal mol- ~for the reaction with Mn 2÷and 3,059 cal mol- ' for the reaction with Mg 2+. For characterization of the 3 enzymes, Km values for substrates and Ki values ofinhibitors were determined. The results of kinetic analysis ofornithine transcarbamylase are consistent with a ping-pong mechanism. For carbamate kinase~ the results of initial velocity studies have indicated that Mn 2÷ADP was as effective a substrate as Mg z÷ADP; and product inhibition studies have shown that the enzyme has 2 distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate, and that its reaction with the substrates is of the random type. The formation of the arginine dihydrolase system in Lactobacilius buchneri NCDOI ~0showed that the specific activities ofarginine deiminase, ornithine transcarbamylase, and carbamate kinase were higher in galactose-grown cells than in glucose - or sucrose-grown cells. The 3 enzymes were induced by arginine, and this enzymatic system was not repressed by glucose. arginine / metabolism / Lactobacillus buchneri I lactic acid bacteria

Introduction The arginine dihydrolase pathway catabolyzes arginine through the action of 3 enzymes:

arginine deiminase (E.C.3.5.3.6), which catalyses the conversion of arginine into citrulline, ornithine transcarbamylase (E.C.2.1.3.3), which is responsible for the transformation of citrul-

ICareer investigator, Consejo Nacional de Investigaciones Cient(lh'as y T~cnicas (CONICET). Argentina. *Author to whom correspondence should be addressed.

M.C. Manca de Nadra et al.

368

line into ornithine and carbamylphosphokinase (E.C.2.7.2.2), which produces ATP from ADP and carbamyl phosphate. NH2

NH~

NH2

H20

HOP02C = NH "~NH3 ~ C = 0 ~-NH2R

I

k

NH-R

NH-R

ADP R

=

(CH2)

3 -

~C=0

I 0 I PO~-

ATP + NH2C00-

CHNH2 - C00H

This pathway has been demonstrated in many bacteria, such as the acetic acid group [1], bacilli [2, 3], Pseudomonas ssp. [4, 5], Clostridia [6, 7], Mycoplasma ssp. [8-10], Aeromonas ssp. [11], Halobacteria [12], Spiroplasma ssp. [13] and Spirochaeta ssp. [14]. In the lactic acid bacteria, evidence of arginine utilization is scant; it has been demonstrated in 2 strains of Streptococcus [15], and there is also a reference to arginine utilization in S. faecium and S. faecalis [16, 17]. The refer~nr.~

In

l.nrtnharilhnc

sate the energy deficit of the cell, thus allowing behavior similar to that of the homofermentative lactobacilli, which obtain 2 mol ATP from 1 mol glucose.

nr~

c A t ~

nr~l;m;n~r~t

data obtained from L.fermentum [18] and those reported by Manca de Nadra et ai. on arginine dihydrolase activity in lactic acid bacteria 119]. They showed that this system is widely distributed in the Betabacteri~Jm /actobacilli; it is quite rare in the Thermobacterium lactobacilli and is absent in the Streptobacterium lactobacilli. The catabolism of arginine via the arginine dihydrolase pathway could be a very important energy-yielding pathway, especially in members of the genus Lactobacil/us in which some of the enzymes of the Krebs cycle appear to be absent. It has been shown that there are strains in the genus Lactobacillus which are unable to grow ~n medium without L-arginine, but there are others which can develop in the absence of this amino acid [20]. Lactobacillus buchneri NCDO110 is heterofermentative, and yields 1 mol ATP per mol glucose consumed. Therefore, to obtain one additional mol ATP from arginine would compen-

Arginine deiminase Lactobacillus buchneri NCDO~ 10(National Culture of Dairy Organisms)does not grow on Larginine as sole energy source, but is able to utilize this amino acid in media supplemented with glucose [21]. Arginase activity was not detected in either whole cells or cell-free extracts of L. bi?chneri NCDO~0. The utilization of arginine by the cells increases growth yield by providing both with an additional growth and energy source [21]. References to arginine deiminase purification and properties are scant [22, 23]. The enzyme has been purified to homogeneity from P. putida [24] and M. arthritidis [25]. In both cases the enzyme is a dimer with a subunit molecular weight of 54,000 from P. putida and 49,000 from M. arthritidis. Arginine deiminase (L-arginine iminohydrolase E.C.3.5.3.3) has been isolated and partially purified in L. buchneri NCDO~0 [26]. The enzyme was purified 68-fold by chromatography on DEAE cellulose, Sepharose 6B and Ultrogel ACA 34 (see T~kl~

1~

The Mr of the enzyme was estimated to be 199,000. The Km value for L-arginine HCI was shown to be 0.83 mM. In P. putida [24], M. arthritidis [25], and S. faeca/is [19], the Km values for L-arginine ranged between 0.1 - 0.4 mM. In L. buchneri [26], L-ornithine, a product of the second enzyme of the arginine dihydrolase pathway, and its analog norvaline act as non-competitive inhibitors with an inhibition consttmt of Ki = 0.55 and 0.22 mM, respectively. The Ki values for ATP and CTP were 0.02 and 0.~ 1 mM, respectively. Neither ADP, GTP nor UTP had any effect on the enzyme. The AG* values of the hydrolysis reaction (8,700 cal mol-~ below 50°C and 380 cal mol-~ above 50°C) indicate that there is a change from one activation energy value to another at the transition temperature. The positive cooperativity observed between Larginine-HCl molecules at pH values different from the optimum (6.0), pointed to the possibility that the protein was oligomeric. The inactivation ofarginine deiminase activ-

Arginine dihydrolase pathway in Lactobacillus buchneri

369

Table I. Purification of arginine deiminase from L. buchneri NCDO~Io. Fraction

Total protein (mg)

Total activity (units)

Cell-free extract (NH4)2 SO4 saturation (50 - 80 %) D E A E - cellulose Sepharose 6B Ultrogel ACA 34 D E A E - cellulose

753.5

1979

2.18

279.0 59.8 42.6 13.8 0.48

1519 1386 1336 781 86

4.50 19.30 26.10 47.20 150.00

Specific activity (units/mg)

Purification (fold) 2.06 8.85 11.97 21.65 68.80

(Printed in: J. AppL Biochem. (1984) 6, 184-187).

ity when p-hydroxymercuribenzoate (PMB) was added to a partially purified enzyme preparation indicates that sulfhydryl groups may be involved at or near the active center of the enzyme. This inactivation by PMB was not suppressed by ethylene-diaminetetraacetic acid (EDTA) or ammonium sulfate, thus indicating that no metallic ion is involved in the structure of the enzyme. Hg 2÷ and Z n 2+ acted as potent inhibitors of arginine deiminase activity. Neither Mg 2÷, Mn 2÷ nor NH~ had any effect on the enzymatic activity. In S.faecalis [221, Mn 2÷produced a 23°/0 inhibition on arginine deiminase activity.

Ornithine transcarbamylase There have been numerous studies on OTCase (carbamylphosphate: L-ornithine carbamyitransferase (E.C.2.1.3.3), but the majority have focused on anabolic OTCase which functions in arginine biosynthesis. The anabolic OTCase is usually repressible by arginine as is the case in E. coli W [27, 28], Salmonella thyphimurium [29] and B. subtilis[30, 31]. Stalone et al. [32,33] established the presence of both a catabolic and an anabolic OTCase in Pseudomonas; they have also shown that the anabolic OTCase of the last organism is unable to catalyze citrulline phosphorolysis. S.faecalis [34] and Mycoplasma [35], which are unable to synthesize arginine. have a single OTCase that only serves a catabolic function. In L. buchneri NCDO~0, Manca de Nadra et al. [36] reported a catabolic OTCase synthesis that was not affected by the presence of arginine. They have demonstrated the presence of only one enzyme with the 2 functions : anabolic and catabolic.

The OTCase from L. buchneri NCDO~0 was partially purified [37]. This purification led to an approximately 14-fold increase in specific activity. The addition of 20% (v/v) glycerol to the buffer stabilized the enzyme in crude cellfree extract and purified enzyme for at least 2 months. In the polyacrylamide gel electrophoresis (Fig. 1) we can see the differences between the fractions obtained in the presence or absence of glycerol. These results on stabilization of enzyme activity are in agreement with those obtained by Powers and Pierson from Neisseria gonorrhoeae [38]. The Mr of the purified enzyme from L. buchneri NCDO~0 was estimated to be ~ 162,000 by gel filtration [37]. This value was smaller than those for Streptococcus ju~,,,,~ ~,~,~s,uuu/ t ~ ' - ' + q , which is a hexamer with a monomeric molecular weight of 39,500, as shown by sodium dodecyl sulfate electrophoresis. B. subtilis has a Mr of 280,000 [30]; however, this value could not be reproduced by Legrain et al. [31], who obtained a Mr of 140,000 for the enzyme from B. subtilis. The catabolic OTCase from Mycoplasma arthritidis has a molecular weight of 360,000, as estimated by sucrose gradient ultracentrifugation [35]. Double reciprocal plots ofornithine and carbamylphosphate saturation curves gave the parallel lines for OTCase of L. buchneri NCDOII0 [37]. The Km values from secondary plots -4 were: Kmcarbamy,phosphate= 7.1 × 10 M and Kmo,nithi,~ ----1.6 × 10-~ M. Dead-end inhibition analysis with norvaline, a structural analog of ornithi/ae, shows that norvaline gave rise to an uncompetitive inhibition pattern when carbamylphosphate was the variable substrate and to a competitive inhibition pattern when ornithine was the variable substrate (Fig.2). These results are consistent with a ping-pong mechanism. The purified OTCase activity of L.

370

M.C. Manca de Nadra et al.

,t a

0 i

I

1

1Iv

:2

[CP mM3-1

2

[Ornithine mM 3-1

b

8-

y. B A

B

C

D

/

2

Fig. 1. Polyacrylamide gel electrophoresis of crude extract (A) and purified ornithine transcarbamylase (B); peak fraction eluted from DEAE-cellulose column chromatography with 20% glycerol (C) and peal: fraction eluted from Ultrogel ACA-34 (D). (Printed in: Current MicrobioL (1984) 11,251-256).

Fig. 2. Dead-end inhibition by norvaline. (a) Reciprocal velocity versus l/[carbamylphosphate] with 3 mM ornithine and 0 (0) and 0.5 (O) mM norvaline. (b) Reciprocal velocity versus l/[ornithine] with 3 mM carbamylphosphate and 0 (0) and 0.5 (©) mM norvaline.

buchneri NCDOi~0 was inhibited by arginine with a Ki = 1.4 x 10-4 M. This result is in agreement with those found by Abdelal et al. [29] from S. thyphimurium. In P. aeruginosa and A. formicans, the catabolic enzyme is induced by arginine [31]. The catabolic enzyme from B. licheniformis is induced by arginine only when 02 is scarce and is repressed by ammonia under these conditions [2, 42]. The arginine dihydrolase pathway was generally induced by arginine and by lack of oxygen [43]. Putrescine, a product ofornithine decarboxylase, inhibited the activity of purified OTCase from L. buchneri NCDOIj0 with a Ki = 7.4 x 10 -3 M [37]. These results are different from those found by

Abdelal et aL [29] from S. typhimutTum. Catabolic OTCase from S.faecalis was not inhibited by putrescine [34]. Stalon et ai. [33] showed that putrescine inhibits the catabolic OTCase from P.fluorescens. The Ki values for other inhibitors of OTCase activity from L. buchneri NCDOII0 were: 1.2 x 10 -4 M for lysine, 1.6 x 10-4 M for ADP, 1.2 × 10-4 M for ATP and 1.4 x 10-4 M for CTP [37]. The optimum pH was between 8.5 9.0, and the positive cooperativeness observed among ornithine molecules at pH values which differed from the optimum pointed to the possibility that the protein was oligomeric. The AG* values of the reactio0 were 9,100 cal molbelow 35°C, and 4,300 cal m o r t above 35"C.

Arginine dihydrolase pathway in Lactobacillus buchneri

371

Carbamate kinase Carbamate kinase catalyzes the reaction ATP + NH3 + CO2 ~ ADP + CAP (CAP = carbamylphosphate), serves to synthesize ATP from carbamylphosphate in those microorganisms able to derive energy from the degradation of guanido compounds, particulary arginine. This reaction proceeds most readily, both kinetically and thermodynamically, toward synthesis of ATP [44-46]. Carbamate kinase (CKase) activity has been reported in Serratia marcesens [47], but Crane and Abdelal [48] reported that adequate criteria were not used to distinguish between carbamate kinase activity and activities of acetate kinase and carbamylphosphate synthase, B. licheniformis [42], B. subtilis [49], Neurospora crassa [50], P. putida [18], Clostridium botulinum [7], Treponema denticola [14], yeast [51], Lactobacillus leichmanii [52] and Lactobacillus buchneri [53]. Frequently however,. CKase activity is assayed only in crude extracts. The enzyme has been purified to homogeneity from S. faecalis [45, 54], Streptococcus faecium [55, 56] and Lactobacillus buchneri [57]. Carbamate kinase of L.buchneri [57] was purified 91-fold from a cell-free extract with apparent homogeneity in PAGE (Fig.3). The molecular weight of the enzyme was found to be 97,000 by polyacrylamide gel electrophoresis. From Streptococcus faecalis [54] CKase was purified 500-fold and appears to be physically homogeneous. From sedimentation studies the apparent molecular weight is ~ 46,000. Marshall and Cohen [45] reported an equivalent molecular weight of 31,000 for CKase from Streptococcus faecalis based on the minimum molecular weight calculated from amino acid composition and on binding of that weight for 1 mol ADP. In Streptococcus faecium (formerly designated as Streptococcus Dr0) [56], the enzyme was purified 26-fold with a molecular weight of 70,000. The molecular weights of CKases from Mycoplasma arthritidis [32] and Pseudomonas aeruginosa [58] were estimated to be 61,000 and 68,000 respectively. T.he CKase of L. buchneri requires M_g2÷ or Mn ~+, the activity being higher with Mn ~+ [57]. The specific activity in the presence of Mg ~+ was 50% lower than that with Mn ~+. The studies on initial velocity of the reverse reaction, with Mg 2÷ or Mn 2+ nucleoside diphosphate as a function of carbamyl phosphate concentration have indicated that phosphoryl group transfer occurs with Mg 2+ ADP or Mn 2+ ADP [59].

I i A

I B

t C

Fig. 3. Different stages ofpolyacrylamide gel electrophoresis fraction patterns during the purification ofcarbamate kinase enzyme. (A) 50-80% ammonium sulfate fraction ; (B) peak fraction eluted on DEAE-cellulose column chromatography; (C) peak fraction eluted on Sepharose 6B column chromatography. (Printed in: Biotechnol. AppL Biochem. (1986) 8, 46-52).

Figures 4a and 4b indicate that the presence of carb+amyl phosphate enhances the binding of Mg ADP or Mn 2÷ ADP and vice versa. It follows then that the reaction has a sequential mechanism in which both the substrates react with the enzyme before either product dissociates, and that the mechanism may be an ordered or random type, since the double reciprocal plots With d~q'erent substrate concentra-

M.C. Manca de Nadra et al.

372

1/v 8 6

Mn2.AD P

a

~

1.0

1/v I 12 -

a

4.0 12.0

10 t ~ 4 . 0 8~ 1 2 . 0

CP - 1.0

I/CPmM 1/v 12 10 8

b

~

1/Mg2*ADPmM

Mg2"ADP 1.0 1/v 4.0 12.0

.:Z .4 .~i .8

8 6

CP

b

1.0 1210

1 1/CPmM

.2 .4 .t5 .8

1 1/Mn2*ADPmM~"

Fig. 4. Effect of Mn 2+ A D ? (a) and Mg 2+ ADP (b) concentration on the initial velocity of reverse reaction with carbamyl phosphate (CP) as the variable substrate. The concentrations of Mg ~+ ADP or Mn ~+ ADP are indicated on the lines of the double-reciprocal plot. (Printed in: Biotechnol. Appl. Biochem. (1987)9, 141-145).

Fig. 5. Effect of carbam~l phosphate (CP) concentration on the initial velocity of reverse reaction with Mg 2÷ ADP (a) or Mn 2÷ ADP (b) as the variable substrate. The CP concentrations are indicated on the lines of the doublereciprocal plot. (Printed in: Biotechnol. Appl. Biochem. 0987) 9, 141-145).

tions display the same form of initial velocity pattern. Figures 5a and 5b show the effect of carbamyl phosphate concentration on the initial reaction velocity with Mg 2+or Mn 2+ ADP as the variable substrate. The Km values for Mg 2+ ADP or Mn 2+ ADP were 0.57 and 0.68 mM respectively, and the values of dissociation constant Ks were 1.51 mM for Mg 2+ ADP and 1.92 mM for Mn 2+ ADP. The Km values for carbamyl phosphate in the presence of Mg 2+ or Mn 2+ ADP were" 1.43 mM and 1.11 mM respectively, and KScarb.,my I phosphate M 2+ ADO --" 1.78 mM; KScarbamy I phosphate 'Mn2+ A D P - - (.66 mM. In CKase of S.faecalis [45], the Km values reported were: Kmcarbam I phos-hate = 100 /zM, and Kmug2+ nDp= 50/1~i In ~'treptococcusfaecalis R [60], the Km values reported were: KmM~2÷At}r = 0.04 mM, and Kmcarb y~ °hosphat~= 114 mM. The Ks values for Mg~+A~P and carbamyl phosphate were" 0.06 mM and 2.0 mM respectively. We have no references concerning the effect of Mn 2÷ on CKase activity in strains of bacteria other than in L.buchneri NCDOj~0.

Mg2+ATP or Mn2+ATP are competitive inhibitors as regards ADP, and non-competitive inhibitors as regards carbamyl phosphate for CKase of L.buchneri NCDO~I0 [59]. The Ki value for competitive product inhibition by Mg2+ATP with Mg~+ADP was 0.9 mM, and the Ki value for non-competitive product inhibition by Mg2+ATP with carbamyi phosphate was 2.0 mM. These patterns of product inhibition would suggest that the enzyme possesses distinct sites for nucleotide and for carbamyl phosphate and that the mechanism of the reaction with the substrates is of random type. These results are in accordance with those reported by Pandey and Pradhan [60] for the reverse reaction of CKase from Streptococcus faecalis. Marshall and Cohen [45] have suggested that the reaction ofcarbamyl phosphokinase with the substrates has an ordered mechanism. Manca de Nadra et al. [,59] reported that the affinity constant for Mn ~+ or Mg ~+ shows that the Km value for Mn 2+ ,(1.53 mM) was lower that the Km value for Mg 2+ (2.56 mM).

Arginine dihydro!ase pathway in Lactobacillus buchneri

Synthesis of arginine dihydrolase pathway enzymes in Lactobacillus buchneri Although considerable detail is available concerning the properties of all enzymes from L. buchneri, the factors affecting enzyme synthesis in bacterial cells are ill-defined. Various i'eports indicate that some streptococci are induced for arginine metabolism when grown on galactose [61, 62], on low concentrations of glucose [63], or in the presence of high arginine concentrations [64, 65]. In Streptococcus iactis strains [66] the specific activities of arginine deiminase and ornithine transcarbamylase were higher when galactose rather than glucose was the growth sugar and arginine was added to the medi.um. In Streptococcusfaecalis [67], the addition of arginine to growing cells results in the coinduction of arginine deiminase, ornithine transcarbamylase, and car-bamate kinase. In Lactobaci/lus leichmanii, Manca de Nadra et al. [68] reported that the specific activities of arginine deiminase and ornithine trancarbamylase were higher when arginine was added to the medium. The formation of the first 2 enzymes of the arginine dihydrolase pathway in this organism seems to be controlled by 2 processes" induction by arginine and repression of the induced synthesis by glucose. In Lactobacillus buchneri [69] the specific activities of the 3 enTymes ¢~ra;n;n,~ ,4,~;_ minase, ornithine transcarbamylase and carbamate kinase) were higher when galactose, rather than glucose or sucrose, was the growth sugar. Crow and Thomas [66] report that in Streptococcus lactis strains, the specific activities of arginine deiminase and ornithine transcarbamvlase were higher when galactose rather than glucose was the growth sugar. In Stret, tococcus faecalis [61] and Streptococcus iactis [62], the enzymes were induced for arginine metabolism when grown on galactose. In L. leichmanii, Manca de Nadra et al. [68] report that the specific activities of arginine deiminase and ornithine transcarbamylase were 2 or 3-fold lower when galactose rather than glucose or lactose was the growth sugar. The enzymes ofL. buchneri are not repressed by glucose [69] and these results in a heterofermentative lactobacillus were different from those obtained with a homofermentative one [68].

373

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