Bioehimica et Biophyiica Acta, 1078 ( 1991 ) 377-382 :f) 1991 Elsevier Science Publishers B.V. 0167-483X/91/'$03 5(I ADO, v-IS O1674838910023,SJ

377

BBAPRO 33957

Distribution and purification of aspartatc raccmase in lactic acid bacteria Hirofumi Okada, Masafumi Yohda, Yuko Giga-Hama, Yukio Ueno, Satoshi Ohdo and Hiromichi Kurnagai Research Center, Asahi Gla,s,i Co., LID., Hazawa-cho, Kanaga~a-ku. Yokahama (Japan~ (Received 18 March 190t)

Key words: Lactic acid bacteria; Aspartate: Racemase: Purificati~m: N-terminal sequence

The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and LactobaciUus species. The enzyme from Streptococcus thermophilus IAMI0064 was more thermostable than that from Streptococcus lactis IAMI198 which contained the enzyme most abundantly a m o n g the lactic acid bacteria we examined here. We p~trified the enzyme about 3400-fold to homogeneity from cell-free extract of S. thermophilus, which is composed of two identical subunits with a molecular weight of 28000 as a homodimer. The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate. Maximal reaction velocity was observed at 37 o C and around pH 8.0. The sequence of the NHz-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-MetGly-Thr-Met.Ala-Thr-Glu-Ser-Phe-.

Introduction

The o-forms of aspartic acid, glutamic acid and alanine are important components in the peptido3lycan layer of bacterial cell walls [1,2]. D-alanine is formed from L-alanine by alanine racemase (EC 5.1.1.1), which is ubiquitously found in bacteria [3,4], whereas oaspartic acid or D-glutamic acid is from a-keto acids (oxalacetate or ot-ketoglutarate) and o-alanine by Damino acid aminotransferasc (EC 2.6.1.21) [5,6]. in several lactic acid bacteria [7,8] which have no D-amino acid aminotransferase activity [9], glutamate racemase plays an important role in providing o-glutamate for the construction of peptidoglycan layer. The presence of aspartate racemase has been demonstrated in Lactobaciils fermenti [10] and Streptococcus faecalis ill]. The aspartatc racemase activity in cell-free extract from L. fermenti was separated by

Corresl:nmdence: H. Okada, Research Cei,ter, Asahi Glass Co.. LTD,, Hazawa-cho, Kanagawa-ku, Yoka~ma 221, Japan.

Sephadex G-100 column chromatography [I0] and purified 60-fold from the supernatant of S. faecalis. The partially purified enzyme, however, showed racemization activity on alanine about half of that on aspartate but no activity on glutamate [11]. Amir'~ acid acemases generally require pyridoxal 5'-phosphate as a cofactor (see Ref. 12 for a review) although some en~mes, such as diaminopimelate epimerase [13] and phenylalanine racemase [14] are independent on added cofactors. Recently, Nakajima and his co-workers found the distribution of glutamate racemase in lactic acid bacteria, purified the enzyme from Pediococcus pentosaceus IF0 3182 [15] and cloned its gene into Escherichia coli [16]. They also found that both enzymes purified from E. coli clone cells and P. pentosaceus do not require cofactors for its catalytic activity [15,16]. Though aspartate racemase had been partially purified from Streptococcus faecalis, the characterization of the enzyme including kinetics and cofactor dependency have not been reported enough [11]. We demonstrated here the purification of asparate racemase from Streptococcus thermophilus IAM10064 and the N-terminal amino acid sequence. We also investigated ~ m e properties of this enzyme.

378 Materials and Methods

Partial purification of aspartate racemase from Streptococcus lactts IAMl198 Streptococcus lactis IAM1198 ceils were anaerobi-

Matenal~

cally grown for 18 h at 37 ° C in 5 I flasks containing 4 1 medium as described above. 9 g wet cells harvested from 8 1 cultures were disrupted for 30 min by a sonic oscillator. The cell debris was removed by centrifugation for 15 rain at 40000 ×g. The resultant cell free extract was applied to DEAE-Sepharose CL-6B column (1.6 x 15 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.2). The active enzyme fraction eluted by a linear gradient of 0 to 0.5 M KCI in 50 mM potassium phosphate buffer (pH 7.2) was collected and applied to Sephacryl S-200 column (1.6 x 60 cm) equilibrated with 50 mM potassium phosphate buffer and eluted with same buffer. The active fractions eluted were collected and used for the study of the enzyme. The enzyme was partially purified about 10-fold with a 20% yield from the cell-free extract.

DEAE-Scpharose CL-6B, DEAE-Scphacel, PhenylSepharos-., Scphacr3q S-300, Sephacryt S-200 and FPLC Mono-Q column and protein molecular weight standards of SDS-PAGE and gel filtration were purchased from Pharmacia (Sweden). D-Amino acid oxidase (hog kidney) was from Sigma (St. Louis, MO, U.S.A.). Chiral pack WH and Crown pack column C R ( + ) were from Daisel Chemical Industries (Osaka, Japan). All other chemicals were purchased from commercial sources.

Culture conditions All strains studied here were cultivated aerobically or anaerobically at 37 °C for about 15 h in a medium (pH 6.0) containing 1.25% polypeptone. 0.55% yeast extract, 1.1% glucose, 1% sodium acet ~te.

Etl2~,me clssov Aspartate racemase activity was determined as follows. o- and L-aspartate in the reaetion mixture was separated by chiral pack WH column (Daisel Chemical Industries) using HPLC after the reaction of the enzyme. The reaction mixture contained 0.75 ~mol of potassium phosphate buffer (pH 8.0) and 2.25 #mol of D- or L-aspartate (pH 8.0 adjusted by NaOH) and aspartate racemase in a final vol. of 100 tH. The reaction was at 37 °C for 2 h and stopped by adding trichloroacetic acid (final concentration of 5%). Amino acids were detected by the fluorescence of reactants with o-phthalaldehyde. Racemization of other amino acids was determined as well, except for glutamic acid using Crown pack column CR( + ). For purification and characterization of the enzyme, aspartate racemase was detected as follows. D-aspartate produced from L-aspartate was converted enzymatically with D-amino acid oxidase; oxalacetate produced by the oxidase reaction from D-aspartate was mixed with 2,4-dinitrophenylbydrazine, as hydrazone was measured. The reaction mixture was composed of 12 p, mol pyrophosphate buffer (pH 8.0), 3 p,mol of L-aspartate, 0.8 mg of D-amino acid oxidase (porcine kidney, Sigma) and aspartate racemase in a final vol of 200/zl. The reaction was done at 37°C for 1 h and added 0.2 ttmol 2,4-dinitrophenylhydrazine containing 2 M HC1, incubated at 37 °C for 30 rain and added 1 M NaOH and the mixture was further incubated at 37°C for 30 rain. The brown color appeared was measured by absorbance at 435 nm with spectrophotometer. Ala~ine racemase activit', was measured with t-alanine as a substrate.

Purification of aspartate racemase from Streptococcus thern,ophilus Cells of S. thermophilus IAMI0064 were grown anaerobically for 18 h on 5 I flasks containing the medium described above. Washed cells from 15 I culture (16 g wet weight) were suspended in 80 rnl of 50 mM potassium phosphate buffer (pH 7.2) containing 1 mM 2-mercaptoethanol and 0.5 mM EDTA (buffer A). All the following purification procedure was carried out at 4 °C except for FPLC at room temperature. The cells were disrupted for 30 rain by a sonic oscillator. Cell debris was removed by centrifugation at 3000 x g for 10 rain. The ,'esultant ceil-free extract was fractionated with ammonium sulfate (35-65% saturation). The active precipitate was dissolved in buffer A and applied to a Phenyi-Sepharose column (1.6 × 15 cm) equilibrated with buffer A containing ammonium sulfate (30% saturation). The enzymc was eluted by a linear gradient of 30 to 0% ammonium sulfate saturation in buffer A. Fractions containing aspartate racemase activity were combined and loaded to Sephacryl S-300 column (1.6 × 65 cm) equilibrated with buffer A. The active enz~ane fractions eluted with buffer A were pooled and applied to DEAE-Sephacel column (1.6 × 15 cm) which had been equilibrated with buffer A. The active fractions eluted with a linear gradient of 0-0.5 M KCI in buffer A were combiued and applied to a Sephacryl S-200 (1.0 × 60 cm) which had been equilibrated with buffer A and eluted with same buffer. The active fractions combined, concentrated by uitrafiltration and then loaded to a Mono-Q column (0.5 × 5 cm) in an FPLC system (Pharmacia, Sweden) and eluted with a linear gradient of KCI from 0 M to 1 M in buffer A. The active fractions were combined and used for further study. Table II ~ummarizes the purification of

379 aspartate racemase. The er=yme was purified about 3400-fold with a 15% yield from the cell-free extract.

TABLE l

l.)l.~tribl~icn ell a~partatc racontzcntopt a~;~ tt?; ft~, /~t~- ~cld boct~cria No activily was h}und in the following ~,tr:lin~: B(ftdohacteriu~.,: b:. fidum t F O 14252. Pedio¢oc('us p~'nto.saccu.~ I F O 38tI1, L,2uconost~a rnesenteroides subsp, m e s e n t e IF() 3426. A , p a r t a l : r a c e m i z a l i o n ac-

Miscellaneous procedures Protein assay was carried out by the Lowry method [17]. Gel filtration was carried out on a Superose 12 H R 10/30 column to estimate the molecular mass of the native form of the enzyme. The enzyme was eluted with buffer A. Albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) were used as markers. The molecular weight of the subunit was determined by polyacrylamide slab-gel electrophoresis in the presence of 0.1% SDS. Phosphorylase b (94) kDa), albumin (67 kDa), ovaibumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and lactoalhttmin (14.4 kDa) were used as markers.

tivity ~ a s d e t e r m i n e d as in M~terials c~nd M e t h o d s 30 t: g protein of c r u d e e×tract from e a c h st~aiv v, as used f~)r assay. Enzq,'me reaction was d o n e for 18 h. ( + ) re~resents 0 - 15e; conversion o f L-aspartate to o-aspartate; ( + + ) 1 5 - 3 0 % : ( 4 ~- ~) 3 0 - 5 0 % . ( - ) indicates no d e t e c t a b l e activity A s p a r l a t e racemiza¢ion activity

Streptococcus faecalis

faegilo11 lactis therrnopttih,s Lactoba:illus bre~ :s bulgaricus casei delbruecktt

Results

Distribution of aspartate racemase in lactic acid bacteria The distribution of aspartate racemase has been reported in two species, i.e. Lactobacillus fermenti [10] and Streptococcus faecalis [11]. Furthermore, incorporation of b-aspartic acid into the peptidoglycan of S. [aecalis and L easel has been reported [18]. These results let us to examine the distribution of the aspartate racemase in lactic acid bacteria including Streptococcus, Lactobacills, Pediocoecus, Leuconostoc and Bifutobacterium species (Table 1). Although the aspartate racemase activity was found in the extract of some species, the activity was too low to detect it quantitatively.

ATCC q7~) I A M 10065 I A M 10067 IAM 1262 IAM 1 198 I A M 10064

+ -* +

IAM IAM IAM IAM

* + + + + + + ~- +

~- * "~ + + +

1318 I 12(,~ 11145 | 085

Table I! showed the results of purification procedure of the enzyme frnm S. thermophilus. The aspartate racemase activities were completely separated from alanine racemase by phenyl-Sepharose column chromatography (Fig. 1). As shown in Table 1I, starting with about 16 g wet cells of S. thermophihts IAM10064, 40 # g of the aspartate raeemase was purified about M00-fold with an overall yield of 15%. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis in the presence of SDS (Fig. 2).

Purification of aspartate racemase As shown in Table I, S. lactis seemed to contain the

Molecular weight and subunit structures

enzyme most abundantly among the lactic acid bacteria examined here. The enzyme from S. lactis, however, was too unstable to purify to homogeneity electrophoretically. We could purify the enzyme to homogeneity from S. therrnophilus because the strain is stable even at 50 o C and include thermostable enzyme.

Apparent molecular weight of the enzyme was 6000(}, as determined by gel filtration column chromatography (Superose 12). The subunit molecular weight was determined to be 28000 by poiyacrylamide gel electrophorcsis iii the presence of SDS (Fig. 2).

TABLE 11 Puriftcation of aspurtare racemase J?om Strept~'occu3 therrnophdus L4M 10004

Cell free extract 3 5 - 6 5 % ( N H 4)2SO, , PhenyI-Sepharose Sephacryl S-300 DEAE-Sephacel Sephacryl S-200 Mono-Q

Protein

Total activity

Yield

Specific acti,¢ity

Purification

(rag)

(/.tmol/min)

(%)

(#mol,/min per mg protein)

(-fold)

914 473 72.6 3.14 4.45 1.62 0.04

7.36 3.77 2.74 3.14 3.20 2.87 1.07

100 51 37 43 43 39 15

0.0118 0 .f~,8 0.038 0.053 0.719 17 8 26.8

1 1 4.8 6.6 90 108 34[~)

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T

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0

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25 II:

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0

50 F.-',ac't ion

i! o"

100

150

number

Fig, 1. PhenyI-Sepharosc column chromatography of a~partate racemase and alanine racema~e from Strt'ptoc'occux thermophih~s IAM 101164, The fraction obtained from 30-60r~ ammorium sulfate precipitate from 16 g of wet cells of S. flu,rmophih~s IAM 10064 was applied. Racemase activity toward aspartate (©) or alanine (e) was a~sayed with 20 #1 of uach fraction. Volume of each fraction. 15 ml (fraction number: I-2IL 111-142), 6 ml (fraction number: 21 - I I~); protein (+): ammonium sut|'ate concentration ( - ).

Enzyme stability Fig. 3 shows the thermal stability of the enzyme purified from S. thermophilus. The residual enzyme activity was measured after the incubation of enzyme at the indicated temperature for 1 h. S. thermophilus enzyme retained its initial activity after the enzyme was treated at 37 ° C for I h in 50 mM potassium phosphate buffer (pH 7.2). S. thermophilus enzyme was also stable even if it was incubated in phosphate buffer between pH 5.5 to 7.5 for 1 h (data not shown). On the othcr hand, p a r t i a l ! y purified enzyme from S. lactis was inactivated completely by thc incubation at 37 ° C for 2

xlO4

\

.J-

30

l

I

i

35

40

/45

~_

50

Temperature ('C) Fig. 3. Thcrmostability of aspartate racemases. Each enzyme (5 n m o l / m i n ) wa~ incubated at indicated temperature for 1 h in 50 mM potassium phosphate buffer (pH 7+2), Aspartate racemase from S. thermophih+s (e)wa~ assayed at 37 ° C and pH 8.0, while the S. lactis t,,a~me (r.) was at 3() + C and pit 7,2.

h. This i:~ the reason why we could not purify the aspartatc racemase from S. lactis.

CTzaracterizat#m of aspartate racemase The optimal condition for aspartate racemase from

S. thermophihLs was at 37 ° C and pH 8.1), whereas that for the enzyme from S. lactis was at 30 ° C and p H 7.2. Fig. 4 shows the activation of aspartate racemase from S. thermophilus by the addition of SH protecting reagent. The enzyme activity in the presence of 2-mercaptoethanol or dithiothreitoi increased 2-fold, as compared that in the absence of it. The enzyme purified from S. thermophih+s did not catalyze the raecmization of alanine and glutamate as a substrate under the condition in which we measured the aspartatc racemase activity as described in Materials and Methods. NH~-terminal amino acid sequence of the enzyme was analyzed by automated Edman de/~radation on Applied Biosystems 470A gas-liquid phase protein sequencer. The sequence obtained was Met-Glu-Asn-

I

150

a

r

I

I --©

120 ~E

~

90

-,~3

z

I

1

0

-ira

6o

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c~

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I

0.5

1

Rf Fig. 2. SDS-polyacrylamide stab-gel eleclrophoresis and molecular mass estimation of purified aspartate racemasc from S. thermopt#lus. 5(~) ng of aspartate racemase was applied. The molecular marker proteins used are listed in Materials and Methods.

30 0

l

10

I

I

I

20

30

40

Concentration

50

(raM)

Fig, 4, Effect of 2-mcrcaptochanol and dithiothreitol on aspartate racemase from S. thermophilus. Partially purified enzyme was assayed in the presence of .-mereaptoethan~ 1( o ) or dithiothreitol (e).

38! Phe-Phe-Ser-lle-keu-Gly-xxx-Met-Gly-Thr-Met-AlaThr-Glu-Ser-Phe-. The 10th amino acid could not be identified.

Discussion In this paper, we described the first purification to homogeneity of aspartate racemase involved in bacterial pcptidoglycan biosynthesis. Staudenbaouer and Strominger reported the incorporation of o-aspartic acid into a peptidoglycan in Streptococcus faecalis and Lactobacills easel via the formation of t)-aspartylphosphate converted by D-aspartic acid activating enzyme [18] and the partial purification of an aspartate racemase from S. facalis ATCC9790 [11]. Although Johnston and Divert succeeded to separate the activity of aspartate racemase from alanine racemase and glutamate raeemase activities in the extracts of L. fermenti, they could net completely purify the enzyme [10]. Until now, properties of the enzyme, such as molecular weight, subunit structure, substrate specificity, cofactor requirement and kinetic parameters have not been clear. Their results led us to study the distribution of aspartate racemase in lactic acid bacteria (Table I). Although Aspartate racemase activity was detected slightly in several strains of Streptococcus and Lactobacillus species (below the order of n m o l / m i n per mg protein), we could obtain 40 # g of the homogeneous enzyme from 16 g of 5, thermophihts in 5 steps with a final yeild of 15% {Table II}. The subunit molecular weight of was calculated to be 28000 by SDS-polyacrylamide gel electrophoresis, whereas native enzyme was eluted with a molecular weight of 60000 from Superose 12 gel filtration column chromatography. Furthermere:, we cloned the aspartate racemase gene by using ;.he DNA probe derived from the N-terminal amino acid sequence and expressed the gene in E. colt. The expressed protein revealed aspartate racemase activity [20]. These results suggested that the enzyme composed of two identical subunits of molecular weight of 28 000. Strominger and his colleagues reported the aspartate racemase fraction obtained from S. faecalis contained the ,~acemization activity for alanine at a half as same as that for aspartate [11]. On the other hand, we demonatrated the complete separation of aspartate racemase activities from alanine racemase by phenylSephamse column chromatography (Fig. 1). As the aspartate racemase purified here had no activity lor racemization of alanine, the enzyme fraction obtained previously from S. faecalis might contain a small amount of alanine racemase. Amino acid racemases generally require pyridoxal 5'-phosphate as a cofactor [t2], although some enzymes such as diaminopimelate epimerase [13] and phenylala-

nine racemase [141 are independent on added cofactors. Recently Nakajima and his colleagues indicated that the purified glutamate racemase o l a lactic acid bacterium, Pediococc,s pe,'ffosaceus, requires no col'actors [15.16]. We found that the addition of pyridoxal 5'-phosphate into the assay mixture showed no effect on the activity of the aspartale racemase from S. thermophilus in the course of purification. We could not determine, however, whether the enzyme might require pyridoxal 5'-phosphate as a cofactm because the yield of the purified enzyme was too low to solve this problem. The cofactor-independent enzymes are thought to contain some sulfhydryl groups essential for activity which acts as a proton-abstracting base in the initial step of amino acid racemase reactions [19]. In the preliminary experiment, the aspartate racemase partially purified from S. thermophilus without 2-mcrcaptoethanol was activated 2-fold by adding sulfhydryl group protecting reagent, such as dithiothreitol or 2mercaptoethanol {Fig. 4). Thiol group might compose of active center on the enzyme and be responsible to catalyze the reaction of racemization. To examine the reaction mechanism of the enzyme including cofactor dependency in detail, we have cloned the gone into E. colt, determined the nucleotide sequence and obtained the enzyme in a quantity from E. coli containing the gene. The results will be described in the following paper.

Acknowledgment We thank Professor Kenji Takahashi, Department of Biophysics and Biochcmistry. Faculty of Science. The Univer.~ity of Tokyo, for helpful advice.

References 1 Perkins, H.R. (1903) Bac~criok Rev. 27. 18. 2 Meiytcr. A. (1965} Biochemist~ of the Amino Acids. Vol. I. Academic Press, New York, pp. l13-11S, 3 Wood. W.A. and Gunsalus. I.e. (1951) J. Biol. Chem. 190. ,1!13. 4 Adamu~. E. (1972} in The Enzymes. Vol. VI (Boyer, PD., ed,L Academic Press. Ne~, York. p. 479. 5 Theme. C B.. Gomez. C.G. and Housewright. R.D (1955} J. Bacteriol. t~9. 357. 6 Yonaha, K.. Misono, H.. Yamamotc~,T. and Soda. K. 11975) J. Biol. Chem. 250. 6983. 7 Narrod. S.A. and Wood. W.A. (1952) Arch. Biochem. Biopkys. 35. 462. 8 Ayengar, P. and Roberts, E. (1952}J. Biol. Chem. 197. 453. 9 Soda, K. and Esaki. N. 11985} Transaminases (Christen. P. and Metzter. D.E.. eds.}, John Wiley& Sons, New York, p. 4~3. 10 Johnston. M.M. and Divert. W.F. (1960) J. Biol. Chem. 240. 5414. t I Lamont. H.C. Slaudenbauer. W.L. and Slrominger. J i . (1972)J. Biol. Chem. 247, 5103. 12 Adams. E. (1976) in Advances in Enzymology.Vol. 44 (Meister. A.. ed.). John Wiley& Sons. New York. p. 69. 13 Wisemar, J.S. and Nichols. J.S. (1984) J. Biok Chore. 250. 8907.

382 14 Yamada, M at~d Kurahashi, K. (1968)J, Biochem. 63, 59. 15 Nakajima, N,, Tanizav, a, K , Tanaka, H. and Soda, K. (1988) Agric. Biol. Chem. 52, 3099. 16 Nakajima, N,, Tanizawa, K.. Tanaka. H. and Soda, K, (1986) ,~gric. Biol. Chem. 50, 2823. 17 L~wry, O.H,, Rosenbraugh, N.J., Farr. AL. and Randall. J. 11951) J, Biol. (,?hem. 193, 265. lg Staude.ba~cr, W. and Strominger, J.L. (1972) J. Biol. Chem. 247, 50Q5

19 Soda, K., Tanaka, H. and Tanizawa, K. (1986) in Vitamin B6, Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects, Part B, (Dolphin, D, Poulson, R, and Avramovic, O., eds.), John Wiley & Sons, New York, p. 223. 20 Yohda, M., Okada, H. and Kumagai, H. (1991) Eiochim Biophys. Aeta, ia press.

Distribution and purification of aspartate racemase in lactic acid bacteria.

The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as...
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