Purification and properties of Aerococcusviridans lactate oxidase

Vol.

164,

No.

October

31,

2, 1989

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1989

PURIFICATION

John

AND

D. Duncan,

Calbiochem, Received

AND

September

10933 18,

919-926

PROPERTIES OF AEROCOCCUS VIRIDANS LACTATE OXIDASE John

0. Wallis,

N. Torrey

Pines

and Mahmood Road,

La Jolla,

R. Azari CA

92037

1989

Lactate oxidase was purified from cells of Aerococcus utilized ammonium viridans by a procedure which sulfate fractionation, DEAE Sepharose CL-6B chromatography, and Sephadex G-100 chromatography. The final preparation was homogeneous by SDS-polyacrylamide gel electrophoresis. The enzyme appears to be a tetramer with a subunit molecular weight of 44,000 and utilizes FMN as a cofactor. The enzyme was highly specific for L-lactate. D-lactate, glycolate, and D,L-2hydroxybutyrate were not oxidized by the enzyme but were competitive inhibitors. The enzyme could be irreversibly inactivated by incubation with bromopyruvate. This inactivation appears to involve a covalent modification near the active site of the enzyme; however, the flavin cofactor is not the site of this modification. a 1983 Rcadem~c Press,Inc.

There are at least two different types of flavin enzymes which oxidize L-lactate and utilize molecular oxygen as the electron acceptor. One of these enzymes catalyzes the overall oxidation and decarboxylation of lactate to acetate and CO2 with the reduction of 02 to H20. This enzyme is produced by Mycobacterium smegmatis and has been well characterized A second type of, lactate oxidase appears to catalyze (1,2,3,4). the oxidation of lactate to pyruvate with the reduction of 02 to This type of lactate oxidase is produced by Tetrahymena H202. pyriformis (51, Streptococcus faecalis (61, and some species of Pediococcus (7,8). The lactate oxidase which is produced by Aerococcus viridans has not been previously characterized but also appears to be of this The purification and some of type. the properties of this enzyme are the subject of this paper. MATERIALS AND METHODS Strain 11563 of American Type Culture

Aerococcus Collection

viridans (Rockville,

was

obtained MD). Meat

from the Peptone-

0006-291x/89 919

$1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in an? form re.rerved.

Vol. 164, No. 2, 1989

‘BIOCHEMICAL

AND BIOPHYSICAL

Type PS was from Marcor Development Amberex 1003 Autolyzed Yeast Extract Corp. (Milwaukee, WI). DEAE Sepharose Sephadex G-200 were obtained from L-lactate (lithium (Uppsala, Sweden). protamine sulfate were from Calbiochem

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Corp. (Hackensack, NJ). was from Universal Foods CL-6B, Sephadex G-100, and Pharmacia Fine Chemicals salt), a-ketobutyrate, and (La Jolla, CA).

was Media and Procedures for Cell Growth. Aerococcus viridans, maintained on Trypticase Soy agar slants which were incubated for cultures were inoculate a 70 ml 48 hr at 30°C. These used to starter culture in a 250 ml baffled flask. The media for the starter culture contained the following: 1) Meat Peptone-Type PS, 16.0 g/l; 2) Amberex 1003 Autolyzed Yeast Extract, 12.0 g/l; 3) Glycerol, 20.0 g/l; 4) K2HP04.3H20, 15.0 g/l; 5) KH2PO4, 5.0 g/l; 10.0 ml/l, 6) Sodium pyruvate, 4.0 g/l; 7) Salt solution, containing MgSO .7H20, 50.0 g/l; FeS04.7H 0, 5.0 g/l; MnC12.4H 0, 3.0 g/l; NaCl, 4 .O g/l; CaC12.2H20, 0.2 g 3 1; ZnS04.7H20, 0.1 g 3 1; Initial pH of the media prior to HCl (concentrated), 2.0 ml/l. autoclaving was adjusted to 7.2 with 5.ON NaOH. The starter culture was placed in an incubator shaker for 20 hr at 30°C and 200 r.p.m. The starter culture was then used to inoculate a fermentor containing 7.0 1 of media having a composition identical to the starter media except for the addition of 0.1 ml/l of Dow P2000 antifoam. The fermentor was operated at 300 r.p.m., 7.0 1.p.m. airflow, and 30°C. The pH was maintained at 7.0 - 7.2 by addition of 25 percent NH40H. Cells were harvested by centrifugation between 24 and 28 hr. Cells were stored frozen until use. Purification of Lactate Oxidase. Cells were resuspended in 2 volumes of 0.05M potassium phosphate buffer (pH 7.0). The suspension was brought to room temperature in a-water bath and 0.5 gm of lysozyme was added per kg of cells. The suspension was then stirred 3 hrs at room temperature. The suspension was chilled to 4OC and the cells were lysed by 3 passes through a Manton-Gaulin homogenizer operating at 6000 psi. The final lysate was diluted 2.5-fold with cold 0.05M potassium phosphate buffer (pH 7.0) and cell debris were removed by centrifugation. Phenylmethylsulfonylfluoride (0.25M in ethanol) was added at a ratio of 1 ml per liter of supernatant. The pH of the supernatant was adjusted to 6.5. Afterwards 14.5 ml of a 2% (w/v) protamine sulfate solution was added per gm in the supernatant. After stirring, a sample was of protein centrifuged and if the resulting supernatant was turbid, Protamine sulfate was additional protamine sulfate was added. centrifugation gave a clear-yellow supernatant. added until Usually 14.5 to 23.5 ml of 2% protamine sulfate was required per gm of protein. The final protamine sulfate supernatant was by ultrafiltration. The pH of the concentrated 3-fold and EDTA was added concentrated preparation was adjusted to 7.0 was added to to a final concentration of 1mM. Ammonium sulfate After 50% saturation and the pH was readjusted to 7.0. centrifugation the precipitate was resuspended in 0.2M KCl, 0.05M potassium phosphate, pH 7 (final volume approximately 10% of the The resuspended concentrated protamine sulfate supernatant). precipitate was clarified by centrifugation and ammonium sulfate to 35% saturation. After centrifugation mOniUm was added sulfate was added to the supernatant to a final concentration of 50% saturation and the pH was adjusted to 7.0. The precipitate was isolated by centrifugation and was resuspended in O.lM KCl, 920

Vol.

164, No. 2, 1989

0.05M potassium same buffer.

BIOCHEMICAL

phosphate

AND BIOPHYSICAL

(pH

7.0)

RESEARCH COMMUNICATIONS

and was dialyzed

against

the

The dialyzed preparation was applied to a column of DEAESepharose CL-6B equilibrated in O.lM KCl, 0.05M potassium phosphate (pH 7.0). Up to 700 units of lactate oxidase were loaded per ml of the ion exchanger. The column was washed with equilibration buffer until protein was not detectable in the effluent. The column was eluted with 0.175M KCl, 0.05M potassium phosphate (pH 7.01, and the yellow fractions were pooled, concentrated, and dialyzed against 0.02M potassium phosphate (pH 7.0). Mannitol (1.3-2 gm/gm protein) was added and the preparation was concentrated by lyophilization. The lyophilized enzyme was stable for up to 1 year when stored desiccated at 4°C. Two hundred mg portions of the lyophilized powder were dissolved in a small volume (l-3 ml) of 0.2M KCl, 0.05M potassium phosphate (pH 7.0) and gel filtered on a 60 x 2.7 cm column of Sephadex G-100 equilibrated in the same buffer. Preparation of Apoenzyme. Lyophilized lactate oxidase which had been purified through the DEAE-Sepharose CL-6B step was resuspended in 0.2M KCl, 0.05M potassium phosphate (pH 7.0). Nine mL of ice-cold O.lM acetic acid, 2.4M ammonium sulfate (final pH 3.6) was added per ml of resuspended lactate oxidase. The preparation was left on ice for 1 hr and was centrifuged. The precipitate was washed twice with lo-ml portions of cold O.lM acetic acid, 2.4M ammonium sulfate. The washed precipitate was resuspended in 0.2M KCl, 0.05M potassium phosphate (pH 7.0). Enzyme Assays. Lactate oxidase activity was determined by a peroxidase-coupled spectrophotometric assay (9). The incubation mixture for the lactate oxidase assay contained the following: 4.5 umol I-aminoantipyrine, 9.8 umol phenol, 30 umol L-lactate 74 us101 magnesium aspartate, 120 umol HEPES, and (lithium salt), 6 units horseradish peroxidase in a final volume of 3 ml and a pH For inhibitor studies the amount of L-lactate was varied of 7.0. from 0.15 umol to 30 umol while the amount of inhibitor was kept fixed at 30 umol. Assays were conducted at 37'C and were started addition of enzyme. Absorbance at 5OOnm was followed. by the defined as the amount of enzyme One unit of enzyme activity is which catalyzes the production of 1 mol H202 per min at 37'C. Molecular Weight Determinations. Molecular weights were determined by SDS polyacrylamide gel electrophoresis and by gel filtration. SDS polyacrylamide gel electrophoresis was performed in gels with 12.6% total acrylamide and 2.7% crosslinker by a procedure similar to that of Laemmli (10). Native molecular weights were determined on a column of Sephadex G-200 phosphate (60 x 2.7 cm) equilibrated in 0.2M KCl, 0.05M potassium (pH 7.0) and on a DuPont GF-250 HPLC column. Protein Determination. determination of protein

The method of Bradford (11) with BSA as the standard.

RESULTS

Lactate described in

oxidase Methods.

AND

was used

for

DISCUSSION

was purified The results 921

of

from this

900 gms purification

of cells as are shown

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164, No. 2, 1969

Table

Purification

1.

of

Lactate

AND BIOPHYSICAL

Oxidase cells

UNITS

STEP

Lysate

BIOCHEMICAL

LACTATE OXIDASE

supernatant

RESEARCH COMMUNICATIONS

from

900 gms of _A. viridans

PROTEIN (gm)

SPECIFIC ACTIVITY (UNITS/mg PROTEIN)

FOLD PURIFICATION

1.70x105

(100%)

48

3.5

Protamine sulfate supernatant

1.49x105

(88%)

17

6.7

1.9

First AMS precipitate (O-SO% sat)

1.38x105

(81%)

5.4

26

7.4

Second AMS precipitate (35-50% sat)

1.06~10~

(62%)

3.0

35

DEAE Sepharose

1.04~10~

(61%)

0.87

Lyophilized

pool

power

7.3x104

10

115

33

272

78

(43%)

Sephadex G-100 poola

Lactate oxidase was purified as described in Methods. Values in parentheses are % yield from lysate supernatant. (AMS, ammonium sulfate). aFor the Sephadex G-100 chromatography 200 mg (4900 unit) of the lyophilized powder was resuspended and chromatographed as described in Methods. 65% of the activity applied to the column was recovered in the pooled fractions.

in Table 1. The lactate oxidase was still quite impure after DEAE-Sepharose CL-6B chromatography. The impurities were removed G-100 and the resulting lactate by chromatography on Sephadex SDS-polyacrylamide oxidase appeared homogeneous by gel electrophoresis (Fig. 1). From this electrophoresis the subunit molecular weight for lactate oxidase was estimated as 44,000. The native molecular weight for lactate oxidase was estimated by gel filtration as From these results it appears that lactate oxidase is a 162,000. tetramer of identical subunits. Several compounds were investigated as potential substrates or inhibitors for lactate oxidase. There was no detectable activity when lactate oxidase was incubated with 1OmM D-lactate, and therefore the enzyme appears to be specific for L-lactate. was no detectable activity when lactate oxidase was There incubated with 1OmM glycolate or 1OmM D,L-2-hydroxybutyrate instead of L-lactate. However, D-lactate, glycolate and D,L-2922

Vol. 164, No. 2, 1989

BIOCHEMICAL

3 Fig.

1.

5

SDS-Polyacrylamide

Samples were

AND BIOPHYSICAL

6

Gel

loaded

RESEARCH COMMUNICATIONS

Electrophoresis.

as follows:

Lane 3, Molecular wt. (94,000; 67,000; 43,000; 30,000; 20,000; Lane 5, Lactate oxidase after DEAE-Sepharose 14,400). step, 6 pg protein. Lane 6, Lactate oxidase after Sephadex G-100 Step, 2 pg protein. markers

competitive inhibitors for the oxidation of hydroxybutyrate were L-lactate as shown in Fig. 2. The Ki values were as follows: glycolate 1.2mM, D-lactate 2.9mM, and 2-hydroxybutyrate 15mM. The Km for L-lactate was 0.43mM. Oxalate and oxamate which are inhibitors for NADH-dependent lactate dehydrogenase (12) and for smegmatis (13) were the lactate monooxygenase from Mycobacterium for A. viridans lactate also tested as potential inhibitors inhibitor (Ki = 5.3mM) but oxidase. Oxalate was a competitive appeared to have no effect over a range of L1OmM oxamate lactate concentration from 0.05 to 5mM (not shown). Incubation of lactate oxidase with bromopyruvate was found to inactivate the enzyme. After 5 hours incubation at room temperature with 20mM bromopyruvate at pH 7.0 usually less than The 2.5% of the initial lactate oxidase activity remained. activity could not be restored by either dilution, extensive Incubation of lactate oxidase with dialysis, or gel filtration. 50mM pyruvate instead of bromopyruvate had no effect on the enzymic activity. Therefore, the inactivation appears to be due to the formation of a covalent adduct between lactate oxidase and 923

Vol.

164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

8

7 7 -6

Fig.

2.

Effects Activity.

I

I

I

I

I

I

I

I

1

2

3

4

5

6

7

8

of

Potential

inhibitors

on

1 9

Lactate

I 10

Oxidase

Incubations were performed as described in Methods at with the varying concentrations of L-Lactate concentration of inhibitor fixed at 10 mM. No addition (m), oxalate (v), glycolate (+), D-lactate (0), D,L 2hydroxybutyrate (0).

Incubation of A L-glycerophosphate bromopyruvate. -t. viridans for 5 hours under similar oxidase with 20mM bromopyruvate conditions resulted in less than a 10% loss in enzymic activity. The effects of competitive inhibitors on the inactivation of The lactate oxidase by bromopyruvate were also investigated. half-live for lactate oxidase incubated with 20mM bromopyruvate at 22OC was 21 min. When the incubation was performed in the presence of 5m.M concentrations of glycolate, D-lactate, or D,Lthe half-live for lactate oxidase was 420 2-hydroxybutyrate, These results suggest min., 69 min., and 53 min., respectively. a mechanism in which bromopyruvate binds to the active site and with an adjacent nucleophile on the enzyme to form a reacts covalent bond. 924

Vol.

BIOCHEMICAL

164, No. 2, 1989

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Experiments were performed to determine if the site for reaction with bromopyruvate was on the apoenzyme or on the flavin. Apoenzymes were prepared from active lactate oxidase, and from lactate oxidase which had been inactivated with bromopyruvate and then dialyzed extensively. The apoenzymes were then reconstituted with either FAD or FMN. Apoenzyme prepared from native lactate oxidase could be almost completely reactivated by addition of FMN but only slightly by the addition of FAD. Therefore, it is likely that FMN is the actual cofactor. For apoenzyme prepared from the bromopyruvate-inactivated lactate oxidase, activity could not be restored by the addition of either FAD or FMN. This indicates that the enzyme had been inactivated by alkylation of the apoenzyme as opposed to alkylation of the FMN cofactor. A. viridans lactate oxidase itself is easily purified, very specific for L-lactate, and appears to be quite stable. In addition, since lactate oxidase is a potential contaminant in preparations of L-glycerophosphate oxidase, selective inhibition of the former by bromopyruvate might have practical applications in clinical chemistry.

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