Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides

Cloning, expressionand characterizationofan extracellular enolasefromLeuconostocmesenteroidesJin-Ha Lee1, Hee-Kyoung Kang1, Young-Hwan Moon2, Dong Lyun Cho3, Doman Kim1,4,Jun-Yong Choe5, R. Honzatko6 & John F. Robyt6

1Engineering Research Institute, Chonnam National University, Gwang-Ju, South Korea; 2Department of Material and Chemical and Biochemical

Engineering, Chonnam National University, Gwang-Ju, South Korea; 3Faculty of Applied Chemical Engineering, Chonnam National University, Gwang-

Ju, South Korea; 4School of Biological Sciences and Technology, Chonnam National University, Gwang-Ju, South Korea and 5California Institute of

Technology, Division of Chemistry, Pasadena, CA, USA; and 6Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University,

Ames, IA, USA

Correspondence: Doman Kim, School of

Biological Sciences and Technology, Chonnam

National University, Gwang-Ju, 500-757,

South Korea. Tel.: 182 62 530 1844;

fax: 182 62 530 0874;

e-mail: [email protected]

Received 4 January 2006; revised 28 March

2006; accepted 7 April 2006.

First published online 3 May 2006.

doi:10.1111/j.1574-6968.2006.00274.x

Editor: Dieter Jahn

Keywords

enolase; Leuconostoc mesenteroides ; cloning;

expression; 2-phospho-D-glucose;

phosphoenolpyruvate.

Abstract

Enolase on the surface of streptococci putatively facilitates pathogenic invasion of

the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpatho-

genic, but it too has an extracellular enolase. Purified isolates of extracellular

dextransucrase from cultures of L. mesenteroides contain minute amounts of

enolase, which separate as small crystals. Expression of L. mesenteroides enolase in

Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing

the conversion of 2-phsopho-D-glycerate to phosphoenolpyruvate. The pH opti-

mum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s�1, respectively. At

phosphate concentrations of 1 mM and below, fluoride is a noncompetitive

inhibitor with respect to 2-phospho-D-glycerate, but in the presence of 20 mM

phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase sig-

nificantly inhibits the activity of purified dextransucrase, and does not bind human

plasminogen. Results here suggest that in some organisms enolase may participate

in protein interactions that have no direct relevance to pathogenic invasion.

Introduction

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) cat-

alyzes the dehydration of 2-phospho-D-glycerate (2PGA) to

phosphoenolpyruvate (PEP) in glycolysis, and the reverse

reaction in gluconeogenesis (Wold & Ballou, 1957; Wold,

1971). All characterized enolases require divalent metal ca-

tions for activity (Wold & Ballou, 1957; Holt & Wold, 1961;

Westhead & McLain, 1964; Wold, 1971; Wang & Himoe, 1974;

Faller et al., 1977; Pietkiewicz & Kustrzeba-Wojcicka, 1983;

Brewer, 1985). The natural cofactor is probably Mg21, which

confers the highest activity (Wold & Ballou, 1957; Faller et al.,

1977; Pietkiewicz & Kustrzeba-Wojcicka, 1983). Enolases are

homodimers in all eukaryotes examined thus far, with native

molecular weights of 80–100 kDa (Wold & Ballou, 1957; Holt

& Wold, 1961; Westhead & McLain, 1964; Wold, 1971; Wang

& Himoe, 1974). Enolases from prokaryotes and archaeons

either are dimers or octamers, the latter with native masses in

the range 350�400 kDa (Kaufmann & Bartholmes, 1992;

Peak et al., 1994; Schurig et al., 1995; Brown et al., 1998a).

Subunit molecular weights of most enolases are c. 45 kDa

(Wold & Ballou, 1957; Holt & Wold, 1961; Westhead &

McLain, 1964; Wold, 1971; Wang & Himoe, 1974; Faller

et al., 1977; Pietkiewicz & Kustrzeba-Wojcicka, 1983; Brewer,

1985; Kaufmann & Bartholmes, 1992; Peak et al., 1994;

Schurig et al., 1995; Brown et al., 1998a). Enolase from

Streptococcus rattus is evidently an exception to the general

trend, being a dimer of native mass 49 kDa (Huther et al.,

1990). Prokaryotic a-enolases are highly conserved proteins

that may function extracellularly in patho-physiological pro-

cesses (Pancholi, 2001). Surface-displayed a-enolase is a major

plasmin(ogen)-binding protein of Streptococcus pneumoniae

(Bergmann et al., 2001). Despite the absence of a signal

sequence and typical motifs required for membrane anchor-

ing, the attachment of enolase (and other glycolytic enzymes)

to cell surfaces of streptococci and pneumococci, and the

attendant effects on plasmin(ogen) acquisition are well estab-

lished (Pancholi & Fischetti, 1998; Bergmann et al., 2001). a-

Enolase (designated Eno) reassociates with the surface of

pneumococci, as observed by immunoelectron microscopy,

with a concomitant increase in plasmin(ogen)-binding capa-

city (Bergmann et al., 2001).

FEMS Microbiol Lett 259 (2006) 240–248c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

In the present study, enolase appears as an impurity in

extracellular preparations of dextransucrase from non-

pathogenic Leuconostoc mesenteroides, an organism used in

the production of clinical dextran and dextran found in

fermentation foods (Kim & Kim, 1999; Kim et al., 2003).

Although present in vanishingly small quantities, enolase

crystallizes from nearly pure preparations of dextransucrase.

To the best of our knowledge, this is the first report of an

extracellular enolase produced by Lactobacillaceae or any

other nonpathogenic prokaryote. In addition, small quan-

tities of recombinant enolase significantly inhibit dextran-

sucrase, suggesting a functional relationship between surface

enolase and dextransucrase in L. mesenteroides.

Materials and methods

Materials

2PGA was purchased from Sigma Chemical Co. (P-0257).

All other chemicals were of reagent grade and commercially

available. Leuconostoc mesenteroides B-512FMCM exhibits a

500-fold increase in extracellular dextransucrase levels rela-

tive to the commercial strain (B-512F) from which it

originated by vacuum UV irradiation and selection (Kim

et al., 1997). Production and secretion of dextransucrase by

strain B-512FMCM is constitutive, whereas dextransucrase

production by strain B-512F is exclusively sucrose inducible.

Restriction endonucleases, alkaline phosphatase and T4

DNA ligase came from Boehringer Mannheim (Mannheim,

Germany), Kosco (Seongnam, Korea) and Takara (Shiga,

Japan), respectively.

Conditions of bacterial culture

Leuconostoc mesenteroides B-512FMCM was grown in LW

medium [0.5% (w/v) yeast extract, 0.5% (w/v) KH2PO4,

0.02% (w/v) MgSO4 � 7H2O, 0.001% (w/v) NaCl, 0.001%

(w/v) FeSO4 � 7H2O, 0.001% (w/v) MnSO4 �H2O, 0.013%

(w/v) CaCl2 � 2H2O] containing 2% glucose at 28 1C without

aeration (Kim et al., 1997; Kim & Kim, 1999).

Preparation of dextransucrase

Isolation of secreted dextransucrase from cultures of L.

mesenteroides followed the procedure of Kitaoka & Robyt

(1998): cells were removed from the medium by centrifuga-

tion (15 000 g, 10 min, 4 1C), and the dextransucrase was

concentrated and dialyzed by passing the bacterium-free

culture through a polysulfone ultrafiltration hollow fiber

cartridge [H5P100-43 (100 kDa cut-off), Amicon, Inc.,

Beverly, MA]. Dialyzed and concentrated dextransucrase

was loaded onto a Toso Haas DEAE 5pw HPLC column,

equilibrated with 100 mM KPi, pH 6.5, and then eluted by a

gradient 0–1 M in NaCl. The eluent was monitored by UV

absorbance at 280 nm. Fractions containing dextransucrase

were verified by the addition of 5 mL of the eluent to 5 mL of

200 mM sucrose, followed shortly thereafter (10 min reac-

tion) by the addition of 30 mL of 100% ethanol. Copious

precipitation of dextran confirmed the presence of dextran-

sucrase. Protein purity was determined by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

(Laemmli, 1997), followed by staining with Coomassie

Brilliant Blue R250 (Laemmli, 1997).

Crystallization of enolase from dextransucrasepreparations

Crystals of extracellular enolase were grown from prepara-

tions of purified dextransucrase by the method of hanging

drops by the combination of 2 mL volumes of protein and

precipitant solutions. The protein solution contained dex-

transucrase (10 mg mL�1), ammonium sulfate (100 mM)

and sodium acetate (50 mM) at pH 5.4. The precipitant

solution contained 24% polyethylene glycol 3000, ammo-

nium sulfate (100 mM), and sodium acetate (50 mM), pH

5.4. Wells contained 500 mL of precipitant solution.

N-terminal amino-acid sequencing

Crystals from several droplets were washed thoroughly with

the precipitation buffer dissolved in de-ionized water and

submitted to the Iowa State University protein facility for N-

terminal sequencing by Edman degradation. Extracellular

proteins isolated from a L. mesenteroides culture were

separated by SDS-PAGE and then transferred electrophor-

etically to a polyvinylidene difluoride (PVDF) membrane

(Bio-Rad, Hercules, CA) (Otter et al., 1987).

DNA isolation and gene cloning

Genomic DNA from L. mesenteroides B-512FMCM was

prepared as described previously (Kim et al., 2000). Routine

DNA manipulations, including plasmid purification and

Escherichia coli transformation, followed protocols of Man-

iatis et al. (1989). Plasmid DNA was isolated from an

overnight culture of Escherichia coli using the alkaline lysis

method (Maniatis et al., 1989). Extraction of chromosomal

DNA and plasmid DNA from agarose gels employed an

AccuPreps Gel Purification kit (Bioneer Co., Daejeon,

Korea). Competent E. coli DH5a cells, prepared by the

procedure of Cohen et al. (1973), were transformed with

plasmid DNA using the CaCl2 method.

Two degenerate primers were designed from the

N-terminal amino-acid sequence, determined in this study,

and from conserved amino-acid sequences for other

bacterial enolases (GenBank accession numbers AF065394,

AAKO04742, AB029313, AJ401152 and AJ303085): E1F, 50-

ATCACTGATATTATCGCACGCGAAGTCCTT-30 and E1R,

FEMS Microbiol Lett 259 (2006) 240–248 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

241Extracellular enolase from Leuconostoc mesenteroides

50-TGAAAGTGAACCAGTTTTGATACCAGC-30. These pri-

mers were used in the amplification of a 1173 bp fragment of

the complete gene. The PCR mixture contained 10 mM Tris/

HCl (pH 8.5), 50 mM KCl, 3 mM MgCl2, 2 mM of each the

deoxyribonucleotide triphosphates (dNTP), 0.25 mg of

genomic DNA from L. mesenteroides B-512FMCM, and

10 pmol of each of the two primers. After incubation for

5 min at 94 1C, 1 mL Taq DNA polymerase (Takara, Japan)

was added, followed by 25 cycles of denaturation (94 1C for

30 s), annealing (62 1C for 30 s) and elongation (72 1C for

30 s). The PCR fragment was ligated into pGEM-T Easy

vector (Promega) for DNA sequencing, followed by the

amplification of 50- and 30-regions by the thermal asym-

metric interlaced (TAIL) PCR method (Liu & Whittier,

1995). The nucleotide sequence of the initial clone facili-

tated the design of internal primers for the forward reaction

(E2R, 50-GCGTTTGCACCCAAGTTACCCTTGTTT-30) and

the backward reaction (E2F, 50-GTTGGTGATGACTT

CTTCGTTACTAACAC-30). Self-ligated L. mesenteroides

B-512FMCM chromosomal DNA, after partial digestion

with BamHI and PstI, was used as a template in PCR,

following the protocols described above. The amplified

fragment was inserted in pGEM-T Easy vector and se-

quenced. Finally, the whole enolase gene was amplified by

PCR using chromosomal DNA from L. mesenteroides B-

512FMCM and two oligonucleotide primers (E1F, as above,

and E3R, 50-TTACTTGTTTTCAATAACTTCG-30) derived

from the nucleotide sequences of the 50- and 30-termini. The

amplified gene was inserted into pGEM-T Easy vector and

cloned using E. coli DH5a.

Over expression and purification ofrecombinant enolase

PCR was performed to introduce a BamHI site at the

initiation codon and a KpnI site downstream from it. The

PCR fragment was ligated into the BamHI and KpnI sites of

the pRSETA vector (Invitrogen, The Netherlands) predi-

gested with BamHI and KpnI. The expression plasmid was

then transformed into E. coli BL21(DE3)pLysS (Invitrogen)

behind the T7 promoter. Escherichia coli BL21(DE3)pLysS

carrying enolA was grown in 50 mL LB medium containing

50mg mL�1 ampicillin and 1 mM isopropyl-a-D-thiogalac-

topyranoside (IPTG) at 28 1C for 6 h. The cells were

harvested by centrifugation (15 000 g, 10 min, 4 1C),

suspended in 200 mL 50 mM imidazole, pH 6.8, and

disrupted by sonication. One percent (v/v) Triton X-100

was added to release the enzyme from the cell membrane.

Cell debris was removed by centrifugation (15 000 g, 10 min,

4 1C). The N-terminal, 6-histidyl tagged (6xHis tag)

protein was purified by nickel-nitrilotriacetic acid-agarose

(Ni-NTA) affinity chromatography (Qiagen, Germany). The

molecular weight of recombinant enolase was determined,

using the Laemmli system (10% w/v) acrylamide gel

(Laemmli, 1997). Proteins were stained with Coomassie

Brilliant Blue R250.

Enolase kinetics

The concentration of recombinant His-tagged enolase was

determined by absorbance of UV radiation at 280 nm, using

an extinction coefficient of 0.9 mL mg�1 cm�1 determined

for rabbit muscle enolase (Kustrzeba-Wojcicka & Golczak,

2002). The activity of recombinant His-tagged enolase was

measured spectrophotometrically at 240 nm by the conver-

sion of 2PGA to phosphoenolpyruvate (Kustrzeba-Wojcicka

& Golczak, 2002). The temperature of assays was 30 1C.

Initial velocities came from the slopes of linear progress

curves of 1 min duration. The assay buffer was 50 mM

imidazole-HCl, pH 6.8, 400 mM KCl and 3 mM MgSO4 in

a total volume of 1.5 mL, unless noted otherwise. Under

these conditions and a substrate concentration of 3 mM, an

increase in absorbance of 0.2 corresponds to the conversion

of 0.226 mmol of substrate (Kustrzeba-Wojcicka & Golczak,

2002). One unit of activity was defined as the conversion of

1 mmol of substrate in 1 min under the described reaction

conditions in the presence of 3 mM 2PGA. In thermostabil-

ity studies, a 10 mL solution of enolase (34.7 U mg�1,

0.003 mg mL�1) was divided into two portions. One sample

was incubated at 50 1C in the assay buffer, with aliquots

drawn at time intervals of 30 min for 3 h. The second sample

was incubated at room temperature as a control. Variations

in enolase activity with respect to pH from 6.0 to 8.3 were

determined by assays using 3 mM 2PGA. The reaction was

initiated by the addition of 10 mL enolase (34.7 U mg�1,

0.003 mg mL�1) to the reaction mixture.

The kinetic constants, Km and kcat, were determined from

measurements of the initial reaction rates, using Linewea-

ver–Burk plots (Lineweaver & Burk, 1934). Concentrations

of 2PGA varied from 0.3 to 5 mM, and the reactions were

initiated by the addition of 10 mL enolase (34.7 U mg�1,

0.003 mg mL�1). The effects of fluoride ions on the kinetics

of enolase from L. mesenteroides B-512FMCM were studied

with and without orthophosphate. Initial reaction rates were

measured in a reaction mixture (1.5 mL) containing 10mL

enolase (34.7 U mg�1, 0.003 mg mL�1), 0.3–5 mM 2-PGA,

0–80 mM fluoride, at 0, 1 and 20 mM phosphate, and

incubated at 30 1C for 1 min. The effects of magnesium, zinc

and manganese divalent cations on enolase activity were

studied. The enzyme solution was dialyzed beforehand

against deionized water, because enzyme prepared in water

does not express any catalytic activity as reported by

Kustrzeba-Wojcicka et al. (1986) and Kustrzeba-Wojcicka

& Golczak (2002). Concentrations of divalent cations varied

from 25 to 1500 mM, containing with 3 mM 2PGA and 10mL

enolase (34.7 U mg�1, 0.003 mg mL�1).


242 J.-H. Lee et al.

Dextransucrase kinetics

The effect of recombinant His-tagged enolase (from

1.49� 10�3 to 14.89� 10�3 nmol per reaction, 1.78 U n-

mol�1) on dextransucrase (8.23� 10�2 nmol, 3.64 U n-

mol�1) was measured by the rate of fructose release from

sucrose (Kim & Kim, 1999). The highly purified dextransu-

crase (8.23� 10�2 nmol, 3.64 U nmol�1) used in these ex-

periments exhibited no detectable enolase activity. The

molecular weight of synthesized dextran was determined

using Bio Gel A-0.5 M (1.5� 100 cm), by the micro redu-

cing value and total carbohydrate analyses (Fox & Robyt,

1991). The degrees of dextranase hydrolysis of dextrans

produced with or without enolase were determined using

Penicillium dextranase (1.0 U; D-4668, Sigma Chemical Co.,

St Louis, MO). The dextranase was added to 1.0 mL,

containing 10 mg of the various dextrans, pH 5.5 (20 mM

citrate-phosphate buffer), at 37 1C and allowed to react

for 3 h. Aliquots (1–5 mL) were added to 20� 20 cm

Whatman K5F TLC plates, which were irrigated at 22 1C,

using two ascents (18 cm path length) of 4 : 10 : 3 volume

proportions of nitromethane–1-propanol–water. The

carbohydrates were visualized on the plate by rapidly

dipping the plate into a solution containing 0.3% (w/v)

N-(1-naphthyl) ethylenediamine and 5% (v/v) H2SO4

in MeOH, dried, and heated at 120 1C for 10 min (Kitaoka

& Robyt, 1998).

Detection of plasminogen binding

To detect plasminogen-binding activity, the recombinant

enolase was incubated overnight at 4 1C with 1%, human

plasminogen (Sigma) in 50 mM Tris-HCl, 110 mM NaCl,

pH 7.4. The enolase–plasminogen mixture was applied to

nickel–nitrilotriacetic acid-agarose (Ni–NTA) affinity col-

umn and checked whether plasminogen was bound to the

recombinant enolase using Chromogenic Assay Kit for

plasma plasminogen (American Diagnostica Inc.).

Results

Characterization of enolase crystals

Isolates of dextransucrase are at least 95% pure based on

SDS-PAGE (data not shown). Such preparations under

conditions of crystallization reproducibly produced tiny

bipyramidal crystals of c. 10–20 mm within 24 h. Protein

from washed crystals provided an unambiguous N-terminal

sequence (SLITDIIARE) that does not exist in the

known sequence of dextransucrase from L. mesenteroides

B-512FMCM, or for that matter, in any know sequence

of dextransucrase. A BLAST search for sequence homology,

using the first 10 residues tentatively identified the crystal-

line material as an enolase. Substantial 2PDG hydrolysis by

dissolved crystals, by the original preparation of dextransu-

crase and by the supernatant of new cultures of L. mesenter-

oides B-512FMCM corroborated the presence of enolase.

Crystals from different preparations of dextransucrase were

used in data collection at the APS-Structural Biology Center,

Argonne National Laboratory, Illinois, and National Syn-

chrotron Light Source, Brookhaven National Laboratory,

New York. All crystals belonged to space group I4 (unit cell

dimensions a = b = 145 A, and c = 101 A) and diffract to

2.4 A resolution. A preliminary structure clearly reveals an

enolase octamer, with two of its subunits defining the

asymmetric unit of the crystal (Jun-Yong Choe and Richard

B. Honzatko, unpublished results).

Cloning and nucleotide sequence of enolase

The 4.3 kb DNA fragment recovered from PCR protocols

and ligated into the BamHI and KpnI sites of the pRSETA

vector is called hereafter pENOLA. The nucleotide sequence

of subclones from pENOLA revealed one major open

reading frame of 1326 bp, coding for a polypeptide of

442 amino-acid residues (Fig. 1). The sequence has been

submitted to GenBank (accession identifier AB088633).

The 442 amino acids encoded by enolA correspond to a

molecular mass of 47 546 Da. The predicted amino-acid

sequence of L. mesenteroides B-512FMCM enolase was

compared with sequences in GenBank using the ClustalW

program (Thompson et al., 1994). ENOLA is similar in

sequence to various bacterial enolases: Lactococcus lactis

ssp. lactis (GenBank accession number, AAK04742)

(Bolotin et al., 2001), 67% identity and 80% homology;

Streptococcus pneumoniae (AJ303085) (Bergmann et al.,

2001), Staphylococcus aureus (AAC17130) (Kuroda et al.,

2001), 62% identity and 75% homology; and Bacillus

subtilis (NP391270), 62% identity and 75% homology

(Kunst et al., 1997).

Purification and characterization ofrecombinant enolase

Purified enolase migrated as a single band on SDS-PAGE,

with a molecular mass of 51.4 kDa (Fig. 2). The difference

between the observed and the calculated mass of 47 546 Da is

largely due to the His-tag. Specific activity of purified

recombinant enolase is 34.7 U mg�1. Enolase activity fell

30% and 36% after 0.5 and 3 h of incubation at 50 1C. The

pH of optimum enolase activity was 6.8 (Fig. 3). Above and

below pH 6.8, the catalytic activity remained constant at

30% maximal activity. Enolase displayed classical Michae-

lis–Menten kinetics. The Km and kcat for 2PGA were

2.61 mM (� 0.01) and 27.53 s�1 (� 1.21), respectively. The

inhibition type of enolase from L. mesenteroides B-

512FMCM by fluoride was noncompetitive with respect to

2PGA in the absence of phosphate (Fig. 4a) and in the



presence of 1 mM phosphate (Fig. 4b), but competitive in

the presence of 20 mM phosphate (Fig. 4c). The Ki for

fluoride ions, calculated using the graphic method of Dixon

& Webb (1979), were 5.4, 4.6 and 9.2 mM in the 0, 1 and

20 mM phosphate, respectively. In the presence of Zn21,

Vmax was 4.11 (� 0.02) mmol min�1 (Table 1).

The addition of enolase to dextransucrase reduced

relative dextransucrase activity by up to 24.9% (Table 2

and Fig. 5) in the presence of 14.89� 10�3 nmol enolase/

8.23� 10�2 nmol dextransucrase (Table 2). As the ratio of

enolase to dextransucrase increased to greater than 0.06, the

relative dextransucrase activity remained much the same

(Fig. 5). The addition of enolase did not significantly change

the structure (branch formation) and polydispersity index

of dextran, based on dextranase hydrolysis and GPC (data

not shown).

Immobilized recombinant enolase did not retain plasmi-

nogen (data not shown), indicating the absence of a high-

affinity interaction between the recombinant enolase and

plasminogen.

Discussion

The sequence of the cloned gene enolA from L. mesenteroides

B-512FMCM is in Fig. 1. The N-terminal region of

Fig. 1. Nucleotide and deduced amino-acid sequences of enolase from

L. mesenteroides B-512FMCM. The N-terminal amino-acid sequence

from the purified L. mesenteroides B-512FMCM enolase crystal is boxed.

Conserved residues with other bacterial enolases (Lactococcus, Strepto-

coccus, Staphylococcus and Bacillus) are printed in boldface.

kDa

200

116

97.4

66.2

45

51.4 kDa

Fig. 2. SDS-PAGE and molecular weight determination of purified

recombinant enolase. The following molecular weight markers (Bio-

Rad) were used: myosin (200 kDa), b-galactosidase (116 kDa), phosphor-

ylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa),

carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme

(14.4 kDa), and aprotinin (6.4 kDa).

0

0.1

0.2

0.3

0.4

5.5 6.5 7.5 8.5pH

2-P

GA

, µM

min

−1

Fig. 3. pH profile of enolase from L. mesenteroides B-512FMCM.



L. mesenteroides B-512FMCM enolase is 75�88% similar to

other bacterial enolases (Lactococcus, Streptococcus, Entero-

coccus and Bacillus). The calculated subunit molecular

weight the encoded protein is 47 546 Da, similar to

subunit molecular weight of the other enolases (Wold

& Ballou, 1957; Pietkiewicz & Kustrzeba-Wojcicka, 1983;

Peak et al., 1994): E. coli enolase 46 kDa (Wold &

Ballou, 1957), Pyrococcus furiousus enolase 45 kDa (Peak

et al., 1994), Saccharomyces cerevisiae enolase 46.7 kDa,

rabbit muscle enolase 41 kDa (Wold & Ballou, 1957) and

carp muscle enolase 49 kDa (Pietkiewicz & Kustrzeba-

Wojcicka, 1983).

Because enolase is a heat shock protein, its high thermo-

stability is not unusual. Similarly, highly thermostable yeast

enolase and Streptococcus rattus (Huther et al., 1990) enolase

have been characterized. Enolases from higher organisms

(carp, rabbit muscle and bovine brain), however, are less

resistant to thermal denaturation (Wold & Ballou, 1957;

Pietkiewicz & Kustrzeba-Wojcicka, 1983; Nazarian et al.,

1992; Kustrzeba-Wojcicka & Golczak, 2002).

0

1

2

3

4

5

−2 −1 0 1 2 3 41/s, mM

1/v

0

2

4

6

8

−1.25 0 1.25 2.5

1/s, mM

1/v

0

1

2

3

4

5

6

7

8

9

−3 −1.5 0 1.5 31/s, mM

1/v

(a)

(b)

(c)

Fig. 4. (a) Determination of the mode for fluoride ions inhibition with-

out phosphate ion. �, 0 mM fluoride ions (SD = � 0.209); ’, 20 mM

fluoride ions (SD = �0.793); m, 40 mM fluoride ions (SD = � 1.516).

(b) Determination of the mode for fluoride ions inhibition in the presence

of 1 mM phosphate ions. �, 0 mM fluoride ions (SD = � 0.256); ’,

20 mM fluoride ions (SD = � 0.702); ., 40 mM fluoride ions (SD =

� 1.622). (c) Determination of the mode for fluoride ions inhibition in

the presence of 20 mM phosphate ions. �, 0 mM fluoride ions (SD =

� 0.209); ’, 20 mM fluoride ions (SD = � 1.352); m, 40 mM fluoride

ions (SD = �1.645).

Table 1. Influence of magnesium, manganese and zinc ions on kinetics

parameters of enolase from L. mesenteroides B-512FMCM

Ion

Vmax (mmol conversion of

2-PGA per min of enzyme) Km (mM)

Mg21 2.91 (�0.02) 0.007 (� 0.001)

Mn21 3.14 (�0.02) 0.009 (� 0.002)

Zn21 4.11 (�0.02) 0.009 (� 0.001)

Parentheses show the standard deviation of Vmax and Km.

PGA, phospho-D-glycerate.

Table 2. Influences of enolase on dextransucrase activity on the average

molecular size and on resistance to dextranase hydrolysis of dextrans

produced

Enolase concentration

in digest

(� 10�3 nmol)�Average

MW (� 106)

Relative

dextransucrase

activity (%)

Unhydrolyzed

dextran

0 95.0 100 7.3

1.49 68.0 59.5 7.7

4.96 67.1 34.6 8.5

9.92 60.4 33.3 8.3

14.89 60.2 24.9 7.5

�Different amounts of the purified enolase (1.78 U nmol�1) were added

on dextransucrase (3.64 U nmol�1) digest.

The enolase activity in the purified dextransucrase was measured and

there was no detectable enolase activity found.

0

20

40

60

80

100

120

0 0.05 0.1 0.15 0.2Enolase/dextransucrase (nmol nmol−1)

Rel

ativ

ede

xtra

nsuc

rase

act

ivity

(%

)

Fig. 5. Influence of enolase on dextransucrase activity.



The pH optima of the enolases from various organisms

are similar. The optimum pH of 6.8 for L. mesenteroides B-

512FMCM enolase is similar to the enolase optima of other

strains (Pietkiewicz & Kustrzeba-Wojcicka, 1983). The pH

activity profile for Leuconostoc enolase seems characteristic

insofar as its activity decreases sharply at pH values above

and below pH 6.8.

The Km value for the enolase from L. mesenteroides

B-512FMCM is 2.61 mM with 2-PGA as substrate, which is

greater than those of other reported enolases, e.g., the Km for

enolase from P. furiousus is 0.38 mM (Peak et al., 1994), carp

enolase Km is 0.31 mM (Pietkiewicz & Kustrzeba-Wojcicka,

1983) and yeast enolase Km is 0.12 mM (Westhead

& McLain, 1964). However, enolase from S. rattus has a Km

of 4.35 mM (Huther et al., 1990). In addition, the specific

activity of L. mesenteroides enolase (34.7 U mg�1) is low

relative to those from other bacterial sources for which

specific activities vary from 100 to 900 U mg�1 (Brown

et al., 1998b). The low specific activity is in part an artifact

due to the use of 3 mM 2PGA as the standard substrate

concentration in the definition of a unit of activity for

enolase. The Vmax value of Fig. 4a translates into a specific

activity (70 U mg�1) that differs only modestly from the

reported range in the literature.

Fluoride is an inhibitor of L. mesenteroides enolase, with

a Ki of 5.4, 4.6 and 9.2 mM in 0, 1 and 20 mM phosphate,

respectively. The Ki values are significantly higher

than inhibition constants for enolases from S. rattus

(Ki�0.85 mM Huther et al., 1990) and from carp muscle

(Ki�0.24 mM Pietkiewicz & Kustrzeba-Wojcicka, 1983) in

the absence of phosphate. Moreover, low concentrations of

phosphate greatly enhance fluoride inhibition of yeast enolase

(Maurer & Nowak, 1981; Nowak & Maurer, 1981). Pi, metal

cations and fluoride combine with the active site to form a

dead-end quaternary complex, which presumably blocks the

productive binding of the substrate in the yeast system.

Unlike the yeast system, fluoride inhibits L. mesenteroides

enolase with no difference in potency or kinetic mechanism

in the absence or presence of phosphate. In a true noncom-

petitive mechanism a fluoride ion must bind to the enzyme-

Mg21-2PGA complex, most likely displacing a water

molecule from the catalytic metal. Charge neutralization

of the catalytic Mg21 by fluoride should destabilize develop-

ing charge on the 3-OH group of 2PGA during the transition

state. The change in mechanism (noncompetitive to com-

petitive) at 20 mM Pi may simply reflect the depletion of

free Mg21 due to the formation of F�-Mg21-Pi complexes

in solution.

For most enolases the most effective metal activator is

Mg21 (Wang & Himoe, 1974; Pietkiewicz & Kustrzeba-

Wojcicka, 1983; Lee & Nowak, 1992a, b). Mg21 strongly

activates enolase from L. mesenteroides B-512FMCM, but

Mn21 and Zn21 are at least equally effective. Mn21 strongly

activates Candida albicans and Saccharomyces cerevisiae

enolases (Lee & Nowak, 1992a, b), but is a weaker activator

of the enolase from carp muscle. Zn21 induces higher

activity than Mn21 in carp enolase, but the converse is true

for the enolases of yeast and C. albicans (Pietkiewicz &

Kustrzeba-Wojcicka, 1983; Lee & Nowak, 1992a, b). In

much of the literature concerning enolases, the metal ion

that binds with highest affinity is called the ‘conformational’

or ‘structural’ metal ion, whereas the metal ion that binds

with lower affinity is the ‘catalytic’ metal ion (Faller et al.,

1977; Brewer, 1985). The activation effect by metal cations

studied here probably relates to metal binding at the

catalytic site of L. mesenteroides enolase.

Streptococcus pneumoniae can colonize the mucosal sur-

faces of the human respiratory tract without clinical symp-

toms (Austrian, 1996). Pneumococci and other Gram-

positive pathogens express specific cell surface components

called adhesins that mediate their adherence to host tissues,

thereby facilitating not only colonization but also invasion

(Cundell et al., 1995). Host plasmin(ogen) binds to cell

surface receptors of these pathogenic bacteria, and at least

two of these receptors are glycolytic enzymes, enolase and

glyceroaldehyde-3-phosphate dehydrogenase (Pancholi &

Fischetti, 1998; Winram & Lottenberg, 1998). Evidently, in

the case of surface enolase from Streptococcus pneumoniae,

a specific sequence (FYDKERKVYD) plays a critical role

in plasmin(ogen) binding (Bergmann et al., 2003). In

L. mesenteroides enolase, the corresponding sequence is

LYDAETKTYK. The absence of a binding interaction between

plasmin(ogen) and L. mesenteroides enolase is consistent with

the suggested critical role played by the central six residues of

the above sequence in the tight binding of synthetic peptides

to plasmin(ogen) (Bergmann et al., 2003).

As L. mesenteroides enolase does not bind plasmin(ogen),

consistent with the nonpathogenic properties of this organ-

ism, then what role does enolase play on the cell surface?

Optimum inhibition of dextransucrase at low molar ratios

of enolase is consistent with two general mechanisms: (i)

Enolase binds to single molecules of dextransucrase, catalyz-

ing a conformational change to a new conformational state

that slowly reverts to its original state after the dissociation

of enolase. (ii) Enolase binds to a dextransucrase multimer,

acting as an allosteric effector. A dextransucrase multimer of

more than a dozen subunits has no support from the

literature. The former mechanism, however, is analogous to

the role of a chaperone, and at least one isozyme of

eukaryotic enolase is a stressed-induced protein presumably

linked to protein folding activities (Aaronson et al., 1995).

Perhaps surface enolase in L. mesenteroides facilitates the

folding of dextransucrase, and dissociates from dextransu-

crase after folding is complete. Clearly protein–protein

interactions involving surface enolases may go well beyond

the recognition of plasmin(ogen).



Acknowledgements

This work was supported by a Korea Research Foundation

Grant (KRF-Y00-290) and National Institutes of Health

Research Grant NS 10546.

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Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides