FEMS Microbiology Letters 79 (1991) 89-94 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100171H

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FEMSLE 04370

Properties of D-lactate dehydrogenase from Lactobacillus bulgaricus." a possible different evolutionary origin

for the D- and L-lactate dehydrogenases

Gis61e Le Bras and J e a n - R e n a u d Gare l

Laboratoire d'Enzymologie du CNRS, 91198 Gif-sur-Yvette, France

Received 19 October 1990 Revision received 22 November 1990

Accepted 23 November 1990

Key words: Lactobacillus bulgaricus; D-Lactate dehydrogenase; Protein evolution

1. SUMMARY

The NAD-dependent D-lactate dehydrogenase from Lactobacillus bulgaricus has been purified to homogeneity. This enzyme was a dimer made of two identical chains of molecular mass 37000. Saturation by either substrate was hyperbolic, with K m values of 50 /~M for N A D H and 1 mM for pyruvate, The specific activity was 2200 uni t s /mg and was not affected by the presence of fructose- 1,6-bisphosphate, Mn 2+ ions, ATP or ADP. The amino-terminal sequence determined on 50 re- sidues showed no significant homology with known lactate dehydrogenases, suggesting that the D- lactate dehydrogenase from L. bulgaricus could not be evolutionarily related to the family of NAD-dependent L-lactate dehydrogenases.

Correspondence to: J.R. Garel, Enzymologie-CNRS, 91198 Gif- sur-Yvette, France.

2. I N T R O D U C T I O N

In many bacteria, lactate is an end-product of fermentation. The reduction of pyruvate by N A D H into the L- or D-isomer of lactate is cata- lyzed by different enzymes, the L-lactate dehydro- genases (L-LDHs) and D-lactate dehydrogenases (D-LDHs). The functional properties of these en- zymes vary widely among bacteria, and from one strain to another in the same genus [1]. Data on amino acid sequences a n d / o r three-dimensional structures of several NAD-dependent bacterial L- LDHs, including several which are allosterically activated by fructose-l ,6-bisphosphate, have shown that they are related to L-LDHs from mammals, birds, and fishes [1-8], and suggest that all L-LDHs belong to the same evolutionary family. Less information is available about the structure of NAD-dependent bacterial D-LDHs. The purification and some properties of the D- LDH from Lactobacillus bulgaricus ( L. delbrueckii

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ssp. bulgaricus) are described in this paper. The N-terminal sequence has been determined on 50 residues and showed no detectable homology with sequences of bacterial or vertebrate L-LDHs, sug- gesting that this D-LDH may have only a distant (if any) evolutionary relationship to the L-LDHs.

3. MATERIALS AND METHODS

3.1. Materials All chemical reagents used were of analytical

grade and were obtained from Merck, except for fructose-l ,6-bisphosphate (Fru-l,6-P2); dithio- threitol (DT-F) and N A D H which were from Sigma. Sepharose CL6B and DEAE-Sephadex A50 were from Pharmacia, and Ultrogel ACA34 was from IBF.

Cells of L. bulgaricus strain B107 were ob- tained from the Centre International de Recherche Daniel Carasso (BSN group), after growth for 4 h in the medium described by De Man et al. [9] (MRS medium from Difco) supplemented with 1% lactose, at 44 o C, the pH being maintained at 6 by the addition of ammonia.

3.2. Purification of D-LDH Bacteria were broken with alumina (about 2 g

alumina per g wet cells) in a buffer composed of 40 mM potassium phosphate, 1 mM MgC12, 0.5 mM EDTA, and 2 mM DTT, pH 6. Nucleic acids were removed by precipitation with 6% (w/v) streptomycine sulphate. After dialysis, the sample was fractionated using differential ammonium sulphate precipitation: D-LDH precipitates be- tween 40 and 60% saturation. The precipitate was collected by centrifugation, dissolved in 20 m M phosphate buffer at pH 6, and dialyzed against t h e same buffer. D-LDH was then loaded on an ion-exchange chromatography co lumn of DEAE- Sephadex A50, and a gradient of ionic strength was applied: D-LDH was eluted between 0.2 and 0.3 M NaCI: After dialysis, the enzyme was sub- jected to gel filtration on an Ul t rogel ACA34 column (80 x 1.2 cm) equilibrated with 40 m M potassium phosphate buffer, 1 mM MgC12, 0.5 mM EDTA, and 2 mM DTT at pH 6. D , L D H was concentrated using either an Amicon Diaflo

cell with a PM10 membrane, or a Centricon 10 microconcentrator, and its homogeneity was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS- PAGE) [10]. The overall yield of purification was higher than 50% in enzyme activity.

Protein concentration was determined by the method of Bradford [11] using immunoglobulins as a standard. The N-terminal amino acid se- quence was determined using an Applied Biosys- terns 470A protein sequencer.

3.3. Measurement of LDH activity LD H activity was measured in 0.4 M potassium

acetate, at pH 5.5 and 27 °C, by the changes in absorbance at 340 nm using an optical path of 0.5 cm. The stereochemical specificity of the enzyme was verified after purification by using either iso- mer of lactate as a substrate for oxidation at alkaline pH [2]. As expected from the properties of L. bulgaricus [1,12], only D-lactate and not L-lactate was converted into pyruvate in 0.1 M glycine buffer at pH 9.5.

4. RESULTS AND DISCUSSION

4.1. Subunit structure of the D-LDH from L. bulgaricus

Homogeneous D-LDH was composed of poly- peptide chains of M r 37 000 as seen from SDS- PAGE (Fig. 1). Gel filtration on a Sepharose CL6B column (120 x 1.2 cm) in the absence or presence of N A D H showed that the apparent molecular mass of D -LD H in native conditions was 75000 (Fig. 2), indicating that the D -LD H from L. bulgaricus is a dimeric protein. This struc- ture was similar to that of D-LDHs from other lactic acid bacteria [1].

4.2. Enzymatic properties of the D-LDH from L. bulgaricus

Purified D-LDH produced 2.2 +__ 0.2 mmol of N A D * per min and per mg of protein when saturated by pyruvate and NADH. This specific activity of 2200 uni t s /mg is one of the highest reported for a purified D-LDH [1].

When determined with 0.4 mM NADH, the saturation curve of D-LDH by pyruvate was hy-

Fig. 1. Homogeneity of the D-L DH from L. bulgaricus as measured by S D S - P A G E on a slab gel containing 10% acrylamide and 0.2% SDS. The gel was stained with Coomassie blue type R and destained by a mixture of 7.5% acetic acid and 30% methanol (v/v) . The molecular mass markers are: phos- phorylase b (M~ 94000), a lbumin (67000), ovalbumin (43000), carbonic anhydrase (30000), and trypsin inhibitor (20 000). The two bands correspond to samples of D-L DH with different amounts of protein, and give a chain molecular mass of 37000

+ 2000.

perbolic at low substrate concentration (Fig. 3) with a K m value of 1 mM. At pyruvate concentra- tions above 8 mM, there was a slight inhibition of the enzyme by an excess of substrate. The satura- tion curve of D - L D H by N A D H was also hyper- boric, with the s a m e K m value of 50 #M for different concentrations of pyruvate (Fig. 4).

In our assay conditions (0.4 M potassium acetate buffer, 7.5 mM pyruvate, 0.4 mM NADH, at pH 5.5 and 27°C), neither the divalent metal cations Mg 2÷ or Mn 2+, nor Fru-l,6-P 2 affected the activity of D-LDH up to a concentration of 5 raM. In addition, D-LDH activity was not in- hibited by ATP, ADP, or GDP up to a concentra-

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1 0 6 .

. . c

1o 5

? N

1 0 4 , ~ • ~ . ~ , ~ ,

0 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 , 8

Partition Coefficient Kav

Fig. 2. Determination of the apparent molecular mass of native D-LDH from L. bulgaricus by gel filtration on a Sepharose CL6B column (120 x 1.2 cm) equilibrated with 20 m M potas- sium phosphate buffer, containing 1 m M MgClz, 0.5 m M EDTA, and 2 m M DTT, at pFl 6, in the presence or absence of 0.4 m M NADH. The standard proteins are: /~-amylase ( M r 200000), alcohol dehydrogenase (150000), serum albumin (67000), ovalbumin (43000), carbonic anhydrase (29000), and cytochrome c (12500). D-LDH was eluted at a position corre-

sponding to a molecular mass of 75 000 + 5000.

tion of 2 mM. In contrast with many other bacteria [1,6,7], the formation of lactate in L. bulgaricus did not seem to be controlled by an allosteric enzyme.

4.3. N-terminal amino acid sequence of the D-LDH from L. bulgarieus

The N-terminal amino acid sequence has been

0 . 8

06

0.4

0,2 ' o : 7

o 1 2 3 4 s

0 ~ , ~,P~-~ , raM-1

5 10 15 20 25

Pyruvate , mM

Fig. 3. Saturation by pyruvate of the D - L D H from L. bulgari- cus determined using 0.4 m M N A D H , in 0.4 M potassium acetate buffer, pH 5.5, at 27°C. Inset: double-reciprocal plot showing that this saturation is hyperbolic up to 4 to 5 m M

pyruvate with a Kr~ value of 1 mM.

92

E

> o8

0.6

0.4

0.2

0 ~-

oL ~7 o 1o 20 3o 40 50 6 0 70 80

I /NADH , raM-1 !

0.1 0.2 0.3 0.4 0.5

N A D H , r n M .

Fig. 4. Saturation by NADH of the D-LDH from L. bulgaricus determined using pyruvate concentrations of 1 mM (©), 7.5 mM (O), or 17 mM (11). Inset: double-reciprocal plot showing

that saturation is hyperbolic with a K m value of 50/.tM,

determined as far as the 50th residue:

NH2- Thr- Lys- I le- Phe-Ala-Tyr- Ala- I le-Arg-Glu-Asp-Glu-Lys-Pro-Phe- Leu-Lys-Glu-Trp-Glu-Asp-Ala-Hi s- Lys-Asp-Val-Glu-Val-Gly-Tyr-Thr- Asp-Lys-Leu-Leu-Thr-Pro-Glu-Asn- X-Ala- Leu- Ala-Lys-Gly- Ala-Asp- Arg-Val-Val-....

A computer-aided search through data banks indi- cated no obvious resemblance with already known protein sequences. The D - L D H from L. bulgaricus is the first D - L D H for which partial sequence information is available, and the above N-terminal sequence is the only that can be used for compari- son with L-LDHs.

The amino-terminal sequences determined over 30 to 40 residues for L-LDHs from mammals, birds, fishes, and from different bacteria belong- ing to different genera such as Bacillus, Bifidobac- terium, Lactobacillus, Thermotoga and Thermus are clearly conserved [2-8], probably because the cor- responding segment is involved in N A D binding [2,4,7]. Several explanations could be proposed for the different N-terminal sequence of L. bulgaricus: (i) the D - L D H from L. bulgaricus is homologous to the L-LDHs, but possesses an extra amino- terminal extension of at least 35-40 residues. This

could still fit with the slightly larger molecular mass of the chain, 37 000 instead of 35 000; (ii) the N A D binding domains of the D - L D H from L. bulgaricus and the L -LDHs are homologous, but this domain does not correspond to the amino- terminal moiety in the D - L D H from L. bulgaricus as in the L-LDHs [2,4,7]; (iii) the D - L D H from L. bulgaricus and the L-LDH family derive from a common ancestor, but have diverged so early that any homology between their sequences is too weak (if any) to be detected; (iv) the D - L D H from L. bulgaricus and the L -LDH family derive from a different ancestor and could then represent a case of convergent evolution. Comparison between the D- and L-LDHs requires further sequence and maybe X-ray crystallography analyses, but the present results are a first indication that these enzymes may not have a simple evolutionary rela- tionship.

A C K N O W L E D G E M E N T S

We are grateful to Akram Fazel, Catherine Duong, and Donna Hartley (Centre International de Recherche Daniel Carasso) for their helpful discussions and supply of material, and to Jean- Pierre Le Ca~r for his excellent determination of the amino-terminal sequence of the protein. This work was supported by Grant nos. LP 2401 from the Centre National de la Recherche Scientifique, 9270300,Biochimie from the Universit6 Paris 6, 87.T.0443 from the Minist+re de la Recherche et de la Technologic, and by the BSN group.

R E F E R E N C E S

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Z. Physiol; Chem. 364,893-909. [4] Hensel, R . Mayr, U. and Yang, C. (1983) Eur. J. Bio-

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[7] Piontek, K., Chakrabarti, P., Sch~ir, H.-P., Rossmann, M.G. and Zuber, H. (1990) Proteins 7, 74-92.

[8] Wrba, A., Jaenicke, R., Huber, R. and Stetter, K.O. (1990) Eur. J. Biochem. 188, 195-201.

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[10] Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. [11] Bradford, M. (1976) Anal. Biochem. 72, 248-254. [12] Kandler, O. and Weiss, N. (1986) in Bergey's Manual of

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