Eur. J . Biochem. lY8, 327-332 (1991) $; FEBS 1991

0014295691003436

Purification, characterization and some properties of diacetyl( acetoin) reductase from Enterobacter aerogenes Javier CARBALLO, Roberto MARTIN, Ana BERNARD0 and Josefa GONZALEZ Laboratory of Food Technology and Biochemistry, Facultad de Veterinaria, Universidad de Leon, Spain

(Received November 26, 1990/February 14, 1991) - EJB 90 1404

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, 0. & Stormer, F. C. (1971) Eur. J. Biochem. 18, 116-1191, for purification of diacetyl(acet0in) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces L-glycols from the corresponding L-a- hydroxycarbonyls by reversible reduction of their 0x0 groups and also reduces the 0x0 group of uncharged a- dicarbonyls converting them into L-a-hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of L-glycol : NAD + oxidoreductase and the recommended name of L-glycol dehydrogenase(NAD).

The molecular mass, PI, affinity for substrates and pH profiles of this enzyme are also described.

The existence of an enzyme capable of reducing diacetyl (butane-2,3-dione) and acetoin (3-hydroxybutan-2-one) was shown for the first time in a wild strain of Enterobacter aerogenes by Bryn et al. [l]), who proposed the name of diacetyl(acet0in) reductase and later proved that the enzyme also accepts pentane-2,3-dione and acetylethyl-carbinol (3- hydroxypentan-Zone) as substrates [2]. Various articles on this enzyme [3 - 61, and on similar enzymes from Klebsiella pneumoniae [7] and Saccaromyces uvarum [8,9], have been published. However, it still has not been classified since sys- tematic studies on its specificity, stereospecificity, reversibility and stoichiometry, necessary for characterization, have not yet been carried out.

This paper describes studies carried out to characterize the diacetyl(acet0in) reductase from E. aerogenes as an L-glycol dehydrogenase(NAD), in our opinion a new oxidoreductase. We also describe here an easy procedure which improves upon that previously described by Bryn et al. [l], and in only three steps gives electrophoretically pure preparations of the above- mentioned enzyme.

MATERIALS AND METHODS Materials

Diacetyl, acetaldehyde, hexanal, acetone, butan-2-one, glyoxal, glyoxylic acid, pyruvic acid, glyceraldehyde, ethanol,

Correspondence to R. M . Sarmiento, Facultad de Veterinaria, Universidad de Leon, Campus de Vegazana, E-24007 Leon, Spain

Abbreviations. [ L Y ] ~ ~ , specific optical rotation for sodium D line at 20 "C.

Enzymes. Diacetyl(acet0in) reductase (EC 1.1.1 .-); diacetyl re- ductase, acetoin : NAD+ oxidoreductase (EC 1 . I .I 3; L-glycol de- hydrogenase, L-glycol : NAD(P)+ oxidoreductase, (EC 1.1.1 .I 85); a-diketone reductase, L-r-hydroxyketone: NAD(P)+ oxidoreductase (EC 1.1 . I . -); alcohol dehydrogenase, Alcohol:NAD+ oxido- reductase (EC 1.1.1 .I); lactate dehydrogenase, (S)-lactate: NAD' oxidoreductase, (EC 1.1 .I .27).

propanol, glycerol and ethyl lactate were obtained from Merck (Darmstadt, Germany); butane-2,3-diol, pentan-3- one, ethyl acetoacetate, butane-1,3-diol and propane-l,2-diol were purchased from Fluka Chemie (Buchs, Switzerland) ; pentane-2,4-dione and hexane-2J-dione were from BDH Chemicals (Poole, England); 2-oxoglutaric acid from F.E.R.O.S.A. (Barcelona, Spain) and pentane-2,3-dione from Eastman (Rochester, NY, USA); methylglyoxal, ethyl pyruvate, methyl pyruvate, glycolaldehyde and a-NADH were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and P-NADPH, P-NADH and P-NAD' from Boehringer (Mannheim, FRG); pentane-2,3-diol and hydroxybutyrate were donated by Dr. Stormer, Oslo University; acetylethylcarbinol was synthesized from hydroxybutyrate using the procedure recommended by Larsen et al. [2]. Acetoin (Fluka Chemie) was purified by the procedure described by Burgos and Martin [lo] to remove traces of diacetyl.

Bacterial strains

As enzyme sources, strains 13048, 15038, 15337, 29007, 29008, 29009,29010,29751 and 29940 of E. aerogenes from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) and strain 2531 from the Czechoslovak Col- lection of Microorganisms (Brno, Czechoslovakia) were tested. ATCC 15038 was finally chosen for purification of the enzyme on the basis of its higher activity.

Methods

Acetoin and butane-2,3-diol were measured by the method of Fuertes et al. [ll], NADH from the A340 ( E = 6220 M- ' .cm-l [12]) and protein by the Bradford procedure [13]. Op- tical rotation was determined using a Perkin Elmer 241 elec- tropolarimeter.

328

Table 1. Purification of diacetyl(acetoin) reductase from E. aerogenes Extracts were obtained from 10 g cell paste. Protein was stimated by the Bradford method [13]. 1 U is the amount of enzyme catalysing the reduction of 1 pmol diacetyl/min

Fraction Total protein Total activity Specific activity Recovery Purification

mg Extract 304 DEAE-cellulose 5.9 Affinity chromatography 2.7

U

1350 8 50 130

4.4 144 270

- - 63 33 54 62

Enzyme assays were carried out at 25"C, by monitoring the change in A340. The reaction mixture contained: 40 pmol substrate, and 0.3 pmol coenzyme and 150 pmol Na2HP04/ KH2P04; total volume, 3 ml, pH 7.0. Oneunit (1 U) is defined as the amount of enzyme that transforms 1 pmol substrate/ min under standard assay conditions, using diacetyl as sub- strate and P-NADH as the coenzyme.

PAGE were carried out using the procedures described by Provecho et al. [14] in 50 mM Na2HP04/KH2P04 buffer (pH 7.8) or 50 mM Tris/borate (pH 9.2). The Fenner method [15] was used to stain protein in gels. Reductase activities in the electrophoresis tubes were revealed as described by Martin and Burgos [16], except for (a) pH of the incubation buffer (pH 7.0), (b) NADH concentration in the incubation mixture (3 mM). To reveal dehydrogenase activities, the method used by Hetland et al. [4] was followed.

SDSjPAGE was carried out using the Webber and Osborn method [17], and electrofocusing by the Ayers and Ericksson procedure [18]. Chromatofocusing was carried out with PBE- 94 gel (Pharmacia), using 25 mM imidazole/HCl (pH 7.4) as starting buffer and polybuffer-74/HCl as eluant.

Enzyme purification

Step 1. Cells were grown in the medium of Davis and Mingioli [19], supplemented with trace elements, as rec- ommended by Kogut and Podoski [20]. After an 11-h incu- bation at 30°C in an orbital shaker (100 strokes/min), cells were collected by centrifugation (2000 x g , 20 min), suspended in distilled water and centrifuged as before at room tempera- ture. All subsequent operations were carried out at 0 - 5 "C.

10 g cells were resuspended in 50 ml 10 mM Na2HP04/ KH2P04 buffer (pH 7.0) and disrupted in a Braun Labsonic 1510 sonicator at 200 W (nine treatments of 15 s). The sonicate was cleared by centrifugation at 40 000 x g for 30 min.

Step 2. The supernatant was chromatographed on a DE- 52 DEAE-cellulose column (2.5 cm x 37 cm) equilibrated and eluted with the buffer used for extracting the enzyme. Elution rate: 190 ml/h; fractions size; 6.2 ml. Enzyme activity was eluted in tubes 17 - 25.

Step. 3. The five best fractions from step 1 were pooled and chromatographed on an NAD-agarose type I column (Pharmacia) of 1 cm x 7 cm, equilibrated with 25 mM mercapto-ethanol in 50 mM Na,HPO,/KH,PO, buffer (pH 7.0). The column was washed with 15 ml 25 mM mercaptoethanol in 50 mM Na2HP04/KH2P04 buffer (pH 7.0) and eluted with 50 ml of a linear gradient of 0- 1.5 mM NAD' in the mixture used for washing (elution rate, 7.5 ml/h). By taking 3-ml fractions, the peak of activity ap- peared around tube 19.

Purified preparations are not stable under freezing, but could be stored for several days at 0 - 5°C with good activity retention.

Fig. I . PAGE at p H 9.2 in 50 mM Tris borate buffer. (A) Stained for protein; (B) stained for diacetyl reductase activity

RESULTS

Purification

Table 1 shows the results of a typical purification exper- iment using the process proposed here.

Electrophoresis of the purified preparations

PAGE at pH 9.2 in Tris/borate buffer (Fig. 1) yielded a single protein band (RF = 0.200 -t 0.007, n = 9) coinciding with activity (RF = 0.199 0.009, n = 6). The same result was obtained by electrophoresis at pH 7.8 in 50 mM Na,HP04/ KH2P04 buffer (protein: RF = 0.140 0.005, n = 7; activity: RF = 0.143 -t 0.004, n = 7).

Stoichiometry

A purified preparation of the enzyme (5 U) was incubated at 25°C with 59.56 pmol NADH and 58.14 pmol acetoin, one of its best substrates. Aliquots were periodically removed and

329

Table 2. Stoichiometry of the reuction Reaction mixture: 58.14 pmol acetoin, 59.56 pmol b-NADH, 5 U enzyme preparation, 2.5 mmol Na2HP04/K2HP04 buffer, pH 7.0; total volume, 50 ml; temperature of incubation, 25°C

Incubation Acetoin NADH butane-2,3-diol time consumed oxidised produced

min pmol

9 18.9 19.4 15.8 16 33.7 33.2 33.4 26 51.7 54.2 55.2 32 57.9 57.2 59.6

acetoin, NADH and butane-2,3-diol were measured. The data in Table 2 show that the enzyme catalyzes reactions of the following type: oxidized substrate + NADH + reduced substrate + NAD' .

Suhstrute specificity The activity in the reductase direction with various alde-

hydes, ketones, hydroxycarbonyls and dicarbonyls was deter- mined using P-NADH (the normal form) as the hydrogen donor. From the results obtained (Table 3), it was deduced that only those compounds which have an 0x0 group close to a hydroxycarbonyl or uncharged carbonyl group are accepted. A single enzyme catalyzes all these reactions. (a) Samples of the second stage of purification were submitted to PAGE at pH 9.2 in Tris/borate buffer and the gels were stained for reductase activity with diacetyl, pentane-2,3-dione, acetoin, ethyl pyruvate, methyl pyruvate and methylglyoxal. A single band was observed in all cases, with an RF = 0.203 k 0.005 (n = 6). (b) Parallel elution profiles were obtained for the six activities in the DEAE-cellulose and NAD-agarose chromato- graphies. (c) Assays with equimolar mixtures of diacetyl and acetoin (13 mM each) gave values lying between those obtained with each of the substrates separately at 13 mM concentrations; similar experiments with mixtures of diacetyl and pentane-2,3-dione, methylglyoxal, ethyl pyruvate or methyl pyruvate gave the same result.

Coenzyme specificity The compounds in Table 3 were also tested as substrates

using P-NADPH or cr-NADH as the hydrogen donor. No measurable activity was obtained.

Stereospecificit-y A purified enzyme preparation containing 1 U activity was

incubated at 2 0 T , in a total volume of 8 ml, with 60.7 pmol fi-NADH and 140 pmol diacetyl. After a 60-min incubation, 57.3 pmol acetoin and a small quantity (1.5 pmol) of butane- 2,3-diol had formed. According to previous data for L-acetoin ([cr];' = f249.1 [21]) and L-butanediol ([cr];' z + I 3 [22]), if the L ( + ) isomers of these products have been formed, the change in [cr];' should be +0.161. A similar value, +0.171, was obtained.

Reversibility Activity tests in the dehydrogenase direction were carried

out with P-NAD' as coenzyme and the following compounds

Table 3. Substrate specificity of diacetyl(acetoin) reductase ,from E. aerogenes. Activity was determined at pH 7.0 in 50 mM Na2HP04/KH2P04 buffer, with a substrate concentration of 13 mM and 0.1 mM b- NADH as coenzyme

Type of substrate Substrate Activity

Monoaldehydes ketone acetaldehyde hexanal acetone butan-2-one pentan-3-one

Uncharged a-dicarbonyl glyoxal methylglyoxal diacetyl methyl pyruvate ethyl pyruvate pentane-2,3-dione

Charged a-dicarbonyl glyoxylic acid pyruvic acid 2-oxoglutaric acid

Non-vicinal dicarbonyl pentane-2,4-dione hexane-2J-dione ethyl acetoacetate

a-h ydroxycarbonyl glycolaldehyde gl yceraldehyde acetoin acet ylethylcarbinol

YO

0 0 0 0 0

trace 11.3

100.0 49.0 51.7 85.6

0 0 0

trace 0 0

0 0

73.1 51.7

as substrates : ethanol, propan-2-01, glycerol, propane-1,2- diol, butane-1,3-diol, butane-2,3-diol, pentane-2,3-diol, gly- colaldehyde, glyceraldehyde, acetoin and ethyl lactate. Only compounds which had two close secondary alcohol groups (butane-2,3-diol and pentane-2,3-diol) were accepted. 1 Unit enzyme oxidized 295 nmol butane-2,3-diol or 151 nmol pen- tane-2.3-diol in 1 min.

Molecular mass and isoelectric point determination The molecular mass of the enzyme, determined by

Sephadex G-200 chromatography (Fig. 2A) turned out to be 61 kDa. SDS/PAGE, after incubating the samples for 2 h at 37 "C in a solution of SDS (1 YO) and 2-mercaptoethanol(l%) at a pH 7.0 in 10 mM Na2HP04/NaH2P04 buffer, yielded a value of 28 kDa (Fig. 2B).

Electrofocusing, in the pH range 3 - 10, of the crude ex- tracts gave a single active band with a maximum at pH 6.8. A similar value (6.85) was obtained by chromatofocusing.

Other properties Fig. 3 shows the effect of pH on the rates of the reactions

catalyzed by this enzyme. The apparent values obtained for V and K , at pH 7.0

in 50 mM Na2HP04/KH2P04 buffer are shown in Table 4. Substrate inhibition was obseved at concentrations of methyl pyruvate above 50 mM or methylglyoxal or ethyl pyruvate over 100 mM.

DISCUSSION Purification

The procedure proposed here for the isolation of the di- acetyl(acetoin) reductase from. E. aerogenes allows for the ob-

330

0.8-

0.6-

> a r 0.4 -

0.2-

A

, I I , I

2 4 10 20 40 100 A Moleculor moss (Dox104)

B

I I I , , ,

2 4 6 8 1 0 100 Moleculor moss (Do x 104) B

Fig. 2. Molecular mass determinations by Sephadeex G-200 chromatog- raphy ( A ) and SDSjPAGE ( B ) . (A) z-globulin, 160 kDa (0); serum albumin, 66.5 kDa (M); egg albumin, 45 kDa ( A ) ; a-chymotrypsin, 24.5 kDa (a); diacetyl(acet0in) reductase (U). (B) serum albumin ( W ) ; egg albumin ( A ) ; myoglobin (0); a-lactalbumin (A); a- chymotrypsin, heavy chain (0 ) ; a-chymotrypsin, light chain (0); diacetyl(acet0in) reductase (g); molecular mass of the reference pro- teins in experiment (B) were taken from [17]

taining of electrophoretically pure preparations with the same specific activity to that of Bryn et al. [l] and improves their method as follows. (a) It gives a better yield, in the region of 50% compared with 20% using the previous method. (b) It uses only very mild fractionation systems, which are not dangerous to the enzyme. (c) It consists of three steps and can be carried out within 24 h, while that of Bryn et al. [I], which includes five precipitations, two dialyses and four chromato- graphies, requires around 10 days. (d) It uses as starting ma- terial a well-characterised bacterial strain, while the previous method uses a wild strain which, as far as we know, is not now available.

Characterization Our results show that the enzyme purified using the strain

ATCC 15038 is the same as that of Bryn et al. [l] isolated

using a wild strain. All the known substrates of the enzyme of Bryn et al. [l] are also used by this enzyme with similar ef- ficiency (compare data in Table 4 with those in [2, 5 , 61). The p l of our enzyme turned out to be 6.8, coinciding with that of one of the most abundant isozymes preparated from the wild strain [4]. Furthermore, the molecular mass of the subunits (28 kDa) agrees well with that of 25-29 kDa obtained by Hetland et al. [3] for the enzyme of Bryn et al. [l]. The main difference between the enzymes is that the one studied here is probably a dimer with a molecular mass of about 60 kDa while that in [l] is a tetramer with a molecular mass close to 100 kDa [3].

The principal issue is, however, how this enzyme should be classified. A considerable number of diacetyl-reducing en- zymes in various biological systems have been described. Un- fortunately, most of the authors who have studied these en- zymes were not interested in characterizing them correctly and usually name them diacetyl reductases with no more investi- gation into their specificity. However, several different en- zymes exist, capable of reducing diacetyl.

Diacetyl reductase (acetoin : NAD + oxidoreductase) first described in Staphylococcus aureus by Strecker and Harary [23]. It is a 68-kDa monomer, very specific for NAD, but accepts all types of a-diketones as substrate (the name a- diketone reductase(NAD) has been recently proposed for it [211).

L - G ~ Y C O ~ dehydrogenase [L-glycol : NAD(P) + oxido- reductase] from hen muscle [24] and bovine liver [14] is a 28 - 30 kDa monomer which presents at least three molecular species of PI 4.8, 6.2 and 7.2. It catalyzes the reduction of uncharged a-dicarbonyls to L-a-hydroxycarbonyls and of these, reversibly, to L-glycols, using NADH or NADPH as hydrogen donor. Sawada et al. [25] have described an enzyme in hamster liver which could also belong to this group.

a-diketone reductase (L-a-hydroxyketone : NAD(P)+ oxi- doreductase) from bovine (141 and pigeon [26] livers reduces a-diketones to L-a-hydroxyketones, as does the enzyme from S. aureus, but accepts NADH or NADPH as coenzyme. The diacetyl reductase from Escherichia coli [27] shows the same substrate and coenzyme specificity, but its stereospecificity has not yet been studied.

Saccharomyces uvarum [28] and Bacillus polymyxa [29] have NADPH-specific diacetyl-reducing enzymes, which have not yet been classified.

NADH-dependent alcohol dehydrogenase [28,30] and lactate dehydrogenase from Staphyloccus epidermidis and S. aureus [21] can also reduce diacetyl in vitro.

The enzyme isolated in this work shows the following properties. It produces L-glycols from the corresponding L-a- hydroxycarbonyls by reversible reduction of their 0x0 groups, and also reduces the 0x0 groups of uncharged a-dicarbonyls, converting them into L-a-hydroxycarbonyls. It is specific for NAD. It does not accept monoaldehydes or pyruvate, thus discarding it as an alcohol or lactate dehydrogenase. It is a 61 kDa dimer with p16.8. These properties clearly differen- tiate it from all the other enzymes able to reduce diacetyl. Its substrate specificity and stereospecificity are similar to that of L-glycol dehydrogenase from animal tissues. However, both enzymes present clear differences in molecular mass and PI and, above all in coenzyme specificity: the enzyme from ani- mal origin can use NADP and NAD, although basically oper- ates with NADP [24, 311, while the enzyme from E. aerogenes only accepts NAD. In our opinion it is a new enzyme for which we propose the recommended name of L-glycol de-

331

100

..-. C .- E . - 60

C

z >

I - .- .- I

Y

20

I I I

' 5 6 7 8 9 10 11 PH

Fig. 3. pHlaciivity profiles of the reactions catalyzed by diacetyljacetoin) reductase from E. aerogenes. ( 0 ) Diacetyl reductase; ( 0 ) pentane- 2,3-dione reductase; ( A ) acetoin reductase; ( W ) ethyl-pyruvate reductase; (0) methyl-pyruvate reductase; (a) methylglyoxal reductase; (A) butane-2,3-diol dehydrogenase. The standard concentrations of substrates and coenzyme were used. Buffers: pH 5.2- 8.4, 50 mM Na2HP04/ KH2P04 ; pH 8.6- 10.6, 50 mM glycine/NaOH

Table 4. Kinetic constants for the reactions catalyzed by diacetyl(acet0in) reductase from E. aerogenes Substrate inhibition was observed at concentrations of methyl pyruvate higher than 50 mM or methylglyoxal or ethyl pyruvate over 100 mM. No inhibition was produced by diacetyl, acetoin, pentane-2,3-dione and buta.ne-2,3-diol at concentrations up to 30 times K,,,

Varied concentration substrate Fixed substrate Apparent K,,, Apparent Vmax VmaxIKn K,

Diacetyl Acetoin Pentane-2,3-dione Methyl glyoxal Ethyl pyruvate Methyl pyruvate Butane-2,3-diol NADH NADH NAD'

1 mM NADH 1 mM NADH 1 mM NADH 1 mM NADH 1 mM NADH 1 mM NADH 1.5 mM NAD'

50 mM diacetyl 10 mM acetoin 50 mM butane-2,3-diol

mM

1.6 0.4 6.0

75 20 18 2.6 0.007 0.005 0.16

pmol/min

4.1 2.7 5.1 2.6 4.6 2.8 2.0

I .min-

2.6 6.8 0.85 0.03 0.23 0.16 0.77

mM

- 204 166 81

hydrogenase(NAD) and the systematic name of L-gly- col : NAD + oxidoreductase.

Kinetic constants The results in Table4 show that diacetyl, acetoin and

butane-2,3-diol are the best accepted compounds. The highest V/Km ratio was obtained with acetoin, which suggests that this may be its physiological substrate. A high V/Km value was also obtained with diacetyl, but this is, in general, far less abundant in biological systems than acetoin.

The enzyme has a high affinity for NADH, with K, values around 5-7 pM. This confirms previous observations [14,

32-34], which state that enzymes which participate in cata- bolic diacetyl reduction should operate in vivo saturated with coenzyme and, consequently, cannot play the role suggested by Burgos and Martin [lo] of regulating the equilibrium be- tween oxidized and reduced forms of pyridine nucleotides.

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