Journal of Dairy Research (1976), 43, 75-83
75
Formation of acetaldehyde from threonine by lactic acid bacteria BY G. J. LEES* Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia AND G. R. JAGO Dairy Research Laboratory, Division of Food Research, C.S.I.R.O., Highett, Victoria 3190, Australia {Received 24 October 1974) SUMMARY. Group N streptococci were found to cleave threonine to form acetaldehyde and glycine. Threonine aldolase, the enzyme catalysing this reaction, was found in all strains except Streptococcus cremoris ZS, an organism which had been shown previously to have a nutritional requirement for glycine. The enzyme was strongly inhibited by glycine and cysteine. The inhibition showed characteristics of allosteric inhibition and was pH-dependent. Inhibition by glycine, but not by cysteine, was highly specific. Analogues and derivatives of cysteine which contained a thiol group and a free amino group inhibited the activity of threonine aldolase. The presence of a carboxyl group was not necessary for inhibition. The cleavage of threonine by wholecell suspensions was stimulated by either an energy source to aid transport, or by rendering the cells permeable to substrate with oleate. Threonine did not appear to be degraded by enzymes other than threonine aldolase, as threonine dehydratase activity was low and NAD- and NADP-dependent threonine dehydrogenases were absent.
Group N streptococci and other lactic acid bacteria produce acetaldehyde during growth in complex media (Bills & Day, 1966; Bottazzi & Dellaglio, 1967; Harvey, 1960; Keenan & Bills, 1968). As described in a previous communication, glucose was found to be a precursor for the acetaldehyde produced by some lactic acid bacteria (Lees & Jago, 1975). Other strains, which produced acetaldehyde during growth but could not form acetaldehyde from glucose, must have utilized some other precursor in the growth medium. Mammalian cells contain the enzyme threonine aldolase which catalyses the cleavage of threonine to acetaldehyde and glycine (Karasek & Greenberg, 1957; Malkin & Greenberg, 1964; Riario-Sforza, Pagani & Marinello, 1969; Shirch & Gross, 1968). The enzyme has been found in only 2 bacteria, Escherichia coli (Lenti & Grillo, 1953) and Clostridium pasteurianum (Dainty, 1967). Threonine was therefore investigated as a possible source of the acetaldehyde produced by lactic acid bacteria during growth. * Present address: Department of Biochemistry, University of Auckland, Auckland, New Zealand.
76
G. J. LEES AND G. R. JAGO MATERIALS AND METHODS
The lactic acid bacteria examined, the growth conditions and the preparation of bacterial cell suspensions and cell-free extracts, have been described previously (Lees & Jago, 1975). Threonine dldolase (E.C. 2.1.2.1) activity was measured by the method of Karasek & Greenberg (1957) except that the Conway microdiffusion units used were size No. 1. The reaction mixture in the outer well of the Conway unit contained: 23 /imoles Na phosphate (NaH 2 PO 4 /Na 2 HPO 4 ),pH7-5; 125 /onoles DL-threonine (pH 7); 0-1 /imoles pyridoxal-5-phosphate (pH 7-0) and 0-25 ml cell-free extract in a total volume of 2 ml. The inner well of the Conway unit contained 1-2 ml of 6-7 mM semicarbazide hydrochloride in 0-2 M Na phosphate, pH 7-0, or 1 ml of distilled water. The acetaldehyde (and ethanol) produced from threonine were estimated by the methods described previously (Lees & Jago, 1976). When acetaldehyde was estimated as the semicarbazone the reaction •was stopped by the addition of 0-25 ml of 20% trichloroacetic acid to the outer well of the Conway unit. When acetaldehyde was estimated by gas chromatography, 0-25 ml of 20% H2SO4 was added to the outer well. Under the conditions used, there was no reduction of acetaldehyde to ethanol or contamination of acetaldehyde by propionaldehyde. All incubations were carried out at 30 °C for 30 min when using cell-free extracts and for 60 min when using whole cell suspensions. Threonine was estimated from the amount of acetaldehyde released after oxidation of threonine by periodate. Stoichiometric amounts of acetaldehyde were released from threonine in concentrations up to 5 /onoles. The sample containing threonine (1 ml), previously boiled for 90 min to remove any residual acetaldehyde, was added to the outer well of a Conway unit. Periodic acid (1 ml of 0-2 M in 0-2 M phosphate buffer, pH 7-0) was added, after which the units were sealed and incubated for 3 h at 30 °C. The acetaldehyde released was collected as the semicarbazone in the centre well and estimated as described previously (Lees & Jago, 1976). Threonine dehydratase (E.C. 4.2.1.16) activity was estimated by the method of Umbarger & Brown (1957), except that the reaction mixtures were boiled for 60 min to remove any acetaldehyde present, before the a-ketobutyrate was estimated with 2,4-dinitrophenylhydrazine. This treatment had no effect on a-ketobutyrate. NAD-dependent threonine dehydrogenase (E.C. 1.1.1.103) was estimated using the reaction mixture of Green & Elliott (1964). The ammo-acetone formed was estimated using acetylacetone (2,4-pentanedione) as the condensing reagent (Mauzerall & Granick, 1956). NADP-dependent threonine dehydrogenase activity was estimated using the reaction mixture of Krauze et al. (1965). The keto acid formed was estimated with 2,4dinitrophenylhydrazine (Friedemann & Haugen, 1943). Protein and dry weight of cells were estimated as described previously (Lees & Jago, 1976). Identification of amino acids. Cell-free extracts of strain DRC3, previously dialysed against 0-1 M-Na phosphate, pH 7-0, were incubated for 16 h in the standard assay mixture which contained either threonine or glycine (62-5 /imoles) plus acetaldehyde (14 or 156 /rnioles). The amino acid products were identified by spotting 5 /A of the
Acetaldehyde from threonine
77
reaction mixtures on plates coated with Silica Gel G and run in 2 solvent systems. 1, upper phase of chloroform:methanol: 17% ammonia (2:1:1 v/v); 2, upper phase of w-butanol:water:acetone:cone, ammonia (200:150:25:25 v/v). The amino-acid spots were developed with ninhydrin. RESULTS
Threonine aldolase activity in cell-free extracts As shown in Table 1, threonine aldolase activity was found in all strains of Group N streptococci examined except Streptococcus cremoris Z8. Threonine aldolase activity also occurred in most of the other lactic acid bacteria examined, but usually at a lower level. Acetaldehyde and glycine, formed from the cleavage of threonine by Str. lactis subsp. diacetylactis DRC3, were identified by gas chromatography (Fig. 1) and thinlayer chromatography (Fig. 2) respectively. In the presence of NADH2, acetaldehyde was reduced to ethanol (Fig. la). The specific activity of threonine aldolase in cell-free extracts of strain DRC3 was not significantly affected when the organism was grown in media containing high concentrations of lactose (5-0%), threonine (0-6%) or glycine (2-0%). However, a concentration of 2 % glycine in the medium strongly inhibited growth (c. 75 % inhibition). The pH at which the cells were grown (Table 2) and the stage of growth at which the cells were harvested had little effect on the levels of threonine aldolase in strain DRC3. Increasing the temperature of growth from 30 to 37 °C, however, decreased the synthesis of threonine aldolase in Str. lactis subsp. diacetylactis and in Str. thermophilus but not in Lactdbacillus bulgaricus (Table 2). Threonine aldolase activity in whole cell suspensions Whole cell suspensions of most of the lactic acid bacteria examined produced greater amounts of acetaldehyde and ethanol from threonine in the presence of glucose as shown in Table 3. Other experiments with L. bulgaricus, which did not produce significant amounts of acetaldehyde or ethanol from glucose alone, showed an increase of up to 58 % in the amount of acetaldehyde produced from threonine when glucose was also present in the reaction mixture. Str. cremoris Z8 and Pediococcus cerevisiae ATCC 8042, which do not have a threonine aldolase, did not show this effect. The addition of small amounts of Na oleate (1 x 10~4 M) to whole cell suspensions greatly stimulated the production of acetaldehyde from threonine (from 2 to 29 /tmoles x 10~4 acetaldehyde produced/min mg (dry wt) cells for Str. lactis subsp. diacetylactis DRC3 and from 10 to 35 /^moles x 10~4 acetaldehyde produced/min mg (dry wt) cells for/Sir. cremoris C13) presumably by increasing the permeability of the bacterial cells (Coles & Lichstein, 1963; Kodicek, 1956). In contrast to the results obtained with whole cell suspensions, with cell-free extracts of Str. lactis subsp. diacetylactis DRC3 a slight inhibition of threonine aldolase activity was obtained with glucose (6% inhibition) or oleate (8% inhibition).
78
G. J. L E E S AND G. R. JAGO
Table 1. Threonine aldolase activities in lactic acid bacteria Organism (A) Group N streptococci
Specific activity!
Streptococcus lactis subsp. diacetylactis DRC1 DRC2
1-29
DRC3 DRC4 18-16
1-56 0-92 0-98 0-37 0-40 0-33 0-59 1-36 0-57 1-28 0-35
Str. lactis
C2 C8F C6 CIO ML3 subsp. maUigenes (MacLeod)
Str. cremoris
HP ML1
0-66 000 0-55 0-59 0-43 0-52 0-40 0-51
E8
Z8 K KH Rl Cl C3 C13
(B) Other lactic acid bacteria Str. faecalis SF1 Str. faecium subsp. durans SD1J Str. thermophilus CSIROJ TW1 ST7
NIZO Lactobacillus bulgariaus%
Leuconostoc cremoris Pediococcus cerevisiae
101
LB1 LB1* LB2* LB3* LB3 LB4*
91404 ATCC 8042*
004 012 0-30 010 0-26 0-33 1-07 0-62 0-69 0-62 0-70 0-70 1-28 001
* Grown in MRS broth. All other organisms grown in tryptone-yeast extract-lactose broth. •(• /tmoles acetaldehyde x 10~2 produced/min mg protein. X Grown at 37 °C. All other organisms grown at 30 °C.
Properties of the threonine aldolase in Str. lactis subsp. diacetylactis DRC3 Under the conditions of assay threonine aldolase activity was linear for at least 60 min. Therefore, an incubation period of 30 min was taken as a measure of the initial reaction velocity. A concentration of L-threonine of 31 mM was found sufficient to saturate the enzyme. The enzyme was found to be specific for the L-isomer of threonine, and the apparent Km value (calculated from a Lineweaver-Burk plot) for the cleavage of this substrate was 3-8 mM. Activity was optimal at pH 7-7 although a relatively high plateau of activity was also present between pH 6-0 and 7-0 (Pig. 3). The stability of the enzyme decreased markedly below pH 5-0 when incubated at 37 °C for 1 h.
Acetaldehyde from threonine (b)
1
79
(c)
16
12
3 2 1f Attenuation: 1 x 16
2 1| 1x16
3
2
1
t
1x8
Retention time, 1 cm = 1 min
Fig. 1. Identification of acetaldehyde, formed from threonine, and its further reduction to othanol in the presence of cell-free extraots of Streptococcus lactis subsp. diacetylactis DRC3. (a) Strain DRC3 + threonine + NADHj, (6) Strain DRC3 + threonine, (c) acetaldehyde(l) + ethanol(2).
Table 2. Effect of temperature and pH of growth on the levels of threonine aldolase in bacterial cell-free extracts Specific activity* after growth at: pH of growth Organism
medium
30 °C
37 CC
Streptococcus lactis subsp. diacetylactis DRC3
NC (final, c. 4-8) 6-3 NC NC NC
1-56
0-78
1-66 111 102 0-40
0-90 107 0-30 003
Str. lactis subsp. diacetylactis DRC3 Lactobacillus bulgaricus LB1 Str. thermophilus CSIRO Escherichia coli K12S
• /(moles acetaldehyde x 10"* produced/min mg protein. All organisms were grown in tryptone-yeastextract broth containing 0-5 % lactose. NC, pH not controlled during growth.
In contrast to the threonine aldolase of Cl. pasteurianum (Dainty, 1967), no requirement for pyridoxal phosphate could be demonstrated for the enzyme in extracts of strain DRC3 dialysed against 0-1 M-Na phosphate buffer, pH 7-0 (to remove pyridoxal phosphate) with or without cysteine. Analogues or derivatives of threonine did not significantly inhibit the total production of carbonyl compounds from threonine. In fact, the production of acetaldehyde from threonine by cell-free extracts was greatly stimulated in the presence of threonyl peptides containing leucine or alanine but not glycine.
80
G. J. L E E S AND G. R. JAGO (a) Solvent front Solvent front
Origin
Origin
Fig. 2. Identification of glyoine as a product of threonine metabolism in Streptococcus laclis subsp. diacetylactis DRC3. (a) 1, Strain DRC3 + water; 2, Strain DRC3 +threonine; 3, Strain DRC3 + glycine + acetaldehyde (156/anole); 4, threonine; 5, glycine + acetaldehyde. Products chromatographed in solvent 1 described under Methods. (6) 1, threonine + glycine; 2, Strain DRC3 + threonine; 3, Strain DRC3 + water; 4, Strain DRC3 + glycine + acetaldehyde (14 /tmole); 5, threonine; 6, allothreonine. Products chromatographed in solvent 2 described under Methods.
Table 3. Stimulation by glucose of the production of acetaldehyde from threonine in bacterial whole cell suspensions (Acetaldehyde + ethanol) • produced from: Organism Streptococcus laclis subsp. diacetylactis DRC3 Str. lactis C2 Str. lactis C8F Str. cremoris HP Str. cremoris Z8 Lactobacillus bulgaricus LB1 Pediococcus cerevisiae ATCC 8042
Glucose 7-02 2-95 0-26
Threonine Threonine + glucose 2-30 4-95 3-56 3-23
11-71 902
5-56
00
018 001
12-95
6-40 25-74 4-93 15-25
o-ot
018
213
1
* /anoles x 10" produced/min mg (dry wt) cells. t Estimated by the semicarbazide method. All other values were estimated by the gas-chromatographic method.
Thiol reagents had little effect on the activity of threonine aldolase. However, thiol compounds containing a free amino group markedly inhibited this activity (Table 4), and as cysteamine inhibited activity a carboxyl group did not appear to be essential for inhibition. Analogues of cysteine which did not contain a thiol group did not
Acetaldehyde from threonine
81
1-2 r-
0-8 -
o E 0.0
04
00 pH
Fig. 3. pH optimum for the production of acetaldehyde from L-threonine by cell-free extracts of Streptococcus lactis subsp. diacetylactis DRC3. The reactions were carried out as described under Methods, using 0' 1 M-N"a phosphate buffers (0) or 0' 1 M-triethanolamine-HCl buffers (O) • The pH values shown refer to the final pHs of the reaction mixtures.
Table 4. Effect of thiol reagents, thiols and analogues of cysteine on the activity of threonine aldolase in cell-free extracts of Str. lactis subsp. diacetylactis DRC3 Compound (A) Thiol reagents —
Iodoacetate .N-ethylmaleimide p-Chloromercuribenzoate (B) Thiola Glutathione (reduced) L-Cysteine D-Cysteine DL-Homocysteine yff-Thio-DL-valine Cysteamine ^-acetyl-L-cysteine y?-thiopropionate a-thioglycerol (C) Analogues of cysteine Cysteic acid L-methionine
Cone, in assay, mM
Belative activity
—
100
0/
/o
11 11 012
98 86 85
25 25 25 25 25 25 25 25 25
88 4 7 3 6 7 85 96 95
25
96
25
94
The reaction mixtures contained DL-threonine (50 mM) as well as the above compounds. The relative activity has been corrected for any interference (less than 3 %) due to carbonyl compounds arising from the thiols or analogues of cysteine.
inhibit. Glutathione or cysteine added to the reaction mixtures did not spontaneously or enzymically reduce the acetaldehyde formed from threonine to ethanol, a possible reason for the non-appearance of acetaldehyde. The threonine aldolase of strain DRC3 was strongly inhibited by cysteine or glycine. Both types of inhibition showed characteristics of allosteric inhibition and were 6
DAR 43
82
G. J. L E E S AND G. R. JAGO 100 r 80 -
10
15
L-cysteine, mM
100 80 ^
60
|
40
ja
£
20 0 10
-20
15
20
25
Glycine, mM
Fig. 4. Inhibition by (a) cysteine and (6) glycine of the threonine aldolase activities in cell-free extracts of Streptoccocus lactis subsp. diacelylactis DRC3: • , pH 6-3; O» pH 7-2; A, pH 7-5; A,pH7-7.
pH-dependent (Fig. 4a, b). Inhibition by glycine was highly specific since alanine, the next higher homologue of glycine, and other analogues such as glyoxylate, ethanolamine, acetamide, taurine and creatine, did not inhibit. However, inhibition by cysteine was not as specific since both cysteine and homocysteine inhibited activity. Alternative pathways for the utilization of threonine did not appear to exist in Str. lactis subsp. diacetylactis DRC3 as NAD- and NADP-dependent threonine dehydrogenases were absent and only low levels of threonine dehydratase (0-07-0-16 x 10~2/tmoles of a-ketobutyrate produced/min mg protein) were present, compared with the relatively high levels of threonine dehydratase (1-56 x 10~2/*moles of aketobutyrate produced/min mg protein) and NAD-dependent threonine dehydrogenase (0-29-0-78 x 10~2 /tmoles of aminoacetone produced/min mg protein) in the control organism, E. coli K12S, grown and assayed under the same conditions. DISCUSSION
The function of threonine aldolase in Group N streptococci may be concerned with the supply of glycine for growth, as Str. cremoris Z8, which did not contain threonine aldolase, has a nutritional requirement for glycine (Reiter & Oram, 1962). The inhibition of threonine aldolase activity by both glycine and cysteine may represent a mechanism by which the biosynthesis of serine from threonine is con-
Acetaldehyde from threonine
83
trolled. It is unknown whether Group N streptococci can form serine from 3-phosphoglyeerie acid. The dependence on pH of the inhibition of threonine aldolase shown by both cysteine and glycine is a property shared by many allosteric enzymes (Cohen, 1965). The rate-limiting step in the cleavage of threonine to acetaldehyde and glycine by whole cells of Group N streptococci would appear to be the transport of threonine into the cell, as glucose stimulated the cleavage of threonine by whole cells, but not by cell-free extracts. Recent work (G. H. Rice & G. R. Jago, unpublished results) has shown that 14C-labelled threonine is actively transported in Str. lactis subsp. diacetylactis DRC3 in the presence of glucose. Threonine aldolase appears to be a major enzyme in the metabolism of threonine by Group N streptococci as a good correlation was observed between the amount of threonine utilized and the amount of acetaldehyde formed by Str. lactis subsp. diacetylactis DRC3, even in the presence of NAD and NADP. Moreover, Str. cremoris Z8, which does not contain a threonine aldolase, did not utilize added threonine. No other enzyme utilizing threonine was found in significant amounts in Str. lactis subsp. diacetylactis DRC3. However, other enzymes involved in the incorporation of threonine into protein and peptidoglycan (Schleifer & Kandler, 1967) may also be present. The low activity of threonine dehydratase may explain, in part, the nutritional requirement of Group N streptococci for isoleucine (Reiter & Oram, 1962), as the formation of a-ketobutyrate is the first step in the bacterial synthesis of isoleucine. This work was supported by grants from the Dairying Research Committee. One of us (G.J.L.) acknowledges the receipt of an Australian Dairy Produce Board Postgraduate Research Studentship and a University of Melbourne Research Scholarship. REFERENCES BILLS, D. D. & DAY, E. A. (1966). Journal of Dairy Science 49, 1473. BOTTAZZI, V. & DELLAGLIO, F. (1967). Journal of Dairy Research 34, 109. COHEN, G. N. (1965). Annual Review of Microbiology 19, 105. COLES, R. S. & LICHSTEIN, H. C. (1963). Archives of Biochemistry and Biophysics 103, 186. DAINTY, R. H. (1967). Biochemical Journal 104, 46P. FRIEDEMANN, T. E. & HAUOEN, G. E. (1943). Journal of Biological Chemistry 147, 415. GREEN, M. L. & ELLIOTT, W. H. (1964). Biochemical Journal 92, 537. HARVEY, R. J. (1960). Journal of Dairy Research 27, 41. KABASEK, M. A. & GKEENBERQ, D. M. (1957). Journal of Biological Chemistry 227, 191. KEENAN, T. W. & BILLS, D. D. (1968). Journal of Dairy Science 51, 1561. KODICEK, E. (1956). In Biochemical Problems of Lipids (2nd international conference, 1953), p. 401. (Eds G. Popjak and E. Le Breton.) London: Butterworths Scientific Publications. KRAUZE, E., KAGAN, Z. S., YAKOVLEVA, V. I. & KRETOVICH, V. L. (1965). Biochemistry (Biokhimiya)
30, 287. LEES, G. J. & JAGO, G. R. (1976). Journal of Dairy Research 43, 63. LENTI, C. & GRILLO, M. A. (1953). Hoppe-Seylers Zeitschrift fur Physiologische Chemie 293, 234. MALKIN, L. I. & GREENBERG, D. M. (1964). Biochimica et Biophysica Acta 85, 117. MAUZERALL, D. & GRANICK, S. (1956). Journal of Biological Chemistry 219, 435. REITER, B. & ORAM, J. D. (1962). Journal of Dairy Research 29, 63. RIARIO-SFORZA, G., PAGANI, R. & MARINELLO, E. (1969). European Journal of Biochemistry 8, 88.
SCHIRCH, L. & GROSS, T. (1968). Journal of Biological Chemistry 243, 5651. SCHLEIFER, K. H. & KANDLER, O. (1967). Archivfiir Mikrobiologie 57, 365. UMBARGER, H. E. & BROWN, B. (1957). Journal of Bacteriology 73, 105. Printed in Oreat Britain 6-2
75
Formation of acetaldehyde from threonine by lactic acid bacteria BY G. J. LEES* Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia AND G. R. JAGO Dairy Research Laboratory, Division of Food Research, C.S.I.R.O., Highett, Victoria 3190, Australia {Received 24 October 1974) SUMMARY. Group N streptococci were found to cleave threonine to form acetaldehyde and glycine. Threonine aldolase, the enzyme catalysing this reaction, was found in all strains except Streptococcus cremoris ZS, an organism which had been shown previously to have a nutritional requirement for glycine. The enzyme was strongly inhibited by glycine and cysteine. The inhibition showed characteristics of allosteric inhibition and was pH-dependent. Inhibition by glycine, but not by cysteine, was highly specific. Analogues and derivatives of cysteine which contained a thiol group and a free amino group inhibited the activity of threonine aldolase. The presence of a carboxyl group was not necessary for inhibition. The cleavage of threonine by wholecell suspensions was stimulated by either an energy source to aid transport, or by rendering the cells permeable to substrate with oleate. Threonine did not appear to be degraded by enzymes other than threonine aldolase, as threonine dehydratase activity was low and NAD- and NADP-dependent threonine dehydrogenases were absent.
Group N streptococci and other lactic acid bacteria produce acetaldehyde during growth in complex media (Bills & Day, 1966; Bottazzi & Dellaglio, 1967; Harvey, 1960; Keenan & Bills, 1968). As described in a previous communication, glucose was found to be a precursor for the acetaldehyde produced by some lactic acid bacteria (Lees & Jago, 1975). Other strains, which produced acetaldehyde during growth but could not form acetaldehyde from glucose, must have utilized some other precursor in the growth medium. Mammalian cells contain the enzyme threonine aldolase which catalyses the cleavage of threonine to acetaldehyde and glycine (Karasek & Greenberg, 1957; Malkin & Greenberg, 1964; Riario-Sforza, Pagani & Marinello, 1969; Shirch & Gross, 1968). The enzyme has been found in only 2 bacteria, Escherichia coli (Lenti & Grillo, 1953) and Clostridium pasteurianum (Dainty, 1967). Threonine was therefore investigated as a possible source of the acetaldehyde produced by lactic acid bacteria during growth. * Present address: Department of Biochemistry, University of Auckland, Auckland, New Zealand.
76
G. J. LEES AND G. R. JAGO MATERIALS AND METHODS
The lactic acid bacteria examined, the growth conditions and the preparation of bacterial cell suspensions and cell-free extracts, have been described previously (Lees & Jago, 1975). Threonine dldolase (E.C. 2.1.2.1) activity was measured by the method of Karasek & Greenberg (1957) except that the Conway microdiffusion units used were size No. 1. The reaction mixture in the outer well of the Conway unit contained: 23 /imoles Na phosphate (NaH 2 PO 4 /Na 2 HPO 4 ),pH7-5; 125 /onoles DL-threonine (pH 7); 0-1 /imoles pyridoxal-5-phosphate (pH 7-0) and 0-25 ml cell-free extract in a total volume of 2 ml. The inner well of the Conway unit contained 1-2 ml of 6-7 mM semicarbazide hydrochloride in 0-2 M Na phosphate, pH 7-0, or 1 ml of distilled water. The acetaldehyde (and ethanol) produced from threonine were estimated by the methods described previously (Lees & Jago, 1976). When acetaldehyde was estimated as the semicarbazone the reaction •was stopped by the addition of 0-25 ml of 20% trichloroacetic acid to the outer well of the Conway unit. When acetaldehyde was estimated by gas chromatography, 0-25 ml of 20% H2SO4 was added to the outer well. Under the conditions used, there was no reduction of acetaldehyde to ethanol or contamination of acetaldehyde by propionaldehyde. All incubations were carried out at 30 °C for 30 min when using cell-free extracts and for 60 min when using whole cell suspensions. Threonine was estimated from the amount of acetaldehyde released after oxidation of threonine by periodate. Stoichiometric amounts of acetaldehyde were released from threonine in concentrations up to 5 /onoles. The sample containing threonine (1 ml), previously boiled for 90 min to remove any residual acetaldehyde, was added to the outer well of a Conway unit. Periodic acid (1 ml of 0-2 M in 0-2 M phosphate buffer, pH 7-0) was added, after which the units were sealed and incubated for 3 h at 30 °C. The acetaldehyde released was collected as the semicarbazone in the centre well and estimated as described previously (Lees & Jago, 1976). Threonine dehydratase (E.C. 4.2.1.16) activity was estimated by the method of Umbarger & Brown (1957), except that the reaction mixtures were boiled for 60 min to remove any acetaldehyde present, before the a-ketobutyrate was estimated with 2,4-dinitrophenylhydrazine. This treatment had no effect on a-ketobutyrate. NAD-dependent threonine dehydrogenase (E.C. 1.1.1.103) was estimated using the reaction mixture of Green & Elliott (1964). The ammo-acetone formed was estimated using acetylacetone (2,4-pentanedione) as the condensing reagent (Mauzerall & Granick, 1956). NADP-dependent threonine dehydrogenase activity was estimated using the reaction mixture of Krauze et al. (1965). The keto acid formed was estimated with 2,4dinitrophenylhydrazine (Friedemann & Haugen, 1943). Protein and dry weight of cells were estimated as described previously (Lees & Jago, 1976). Identification of amino acids. Cell-free extracts of strain DRC3, previously dialysed against 0-1 M-Na phosphate, pH 7-0, were incubated for 16 h in the standard assay mixture which contained either threonine or glycine (62-5 /imoles) plus acetaldehyde (14 or 156 /rnioles). The amino acid products were identified by spotting 5 /A of the
Acetaldehyde from threonine
77
reaction mixtures on plates coated with Silica Gel G and run in 2 solvent systems. 1, upper phase of chloroform:methanol: 17% ammonia (2:1:1 v/v); 2, upper phase of w-butanol:water:acetone:cone, ammonia (200:150:25:25 v/v). The amino-acid spots were developed with ninhydrin. RESULTS
Threonine aldolase activity in cell-free extracts As shown in Table 1, threonine aldolase activity was found in all strains of Group N streptococci examined except Streptococcus cremoris Z8. Threonine aldolase activity also occurred in most of the other lactic acid bacteria examined, but usually at a lower level. Acetaldehyde and glycine, formed from the cleavage of threonine by Str. lactis subsp. diacetylactis DRC3, were identified by gas chromatography (Fig. 1) and thinlayer chromatography (Fig. 2) respectively. In the presence of NADH2, acetaldehyde was reduced to ethanol (Fig. la). The specific activity of threonine aldolase in cell-free extracts of strain DRC3 was not significantly affected when the organism was grown in media containing high concentrations of lactose (5-0%), threonine (0-6%) or glycine (2-0%). However, a concentration of 2 % glycine in the medium strongly inhibited growth (c. 75 % inhibition). The pH at which the cells were grown (Table 2) and the stage of growth at which the cells were harvested had little effect on the levels of threonine aldolase in strain DRC3. Increasing the temperature of growth from 30 to 37 °C, however, decreased the synthesis of threonine aldolase in Str. lactis subsp. diacetylactis and in Str. thermophilus but not in Lactdbacillus bulgaricus (Table 2). Threonine aldolase activity in whole cell suspensions Whole cell suspensions of most of the lactic acid bacteria examined produced greater amounts of acetaldehyde and ethanol from threonine in the presence of glucose as shown in Table 3. Other experiments with L. bulgaricus, which did not produce significant amounts of acetaldehyde or ethanol from glucose alone, showed an increase of up to 58 % in the amount of acetaldehyde produced from threonine when glucose was also present in the reaction mixture. Str. cremoris Z8 and Pediococcus cerevisiae ATCC 8042, which do not have a threonine aldolase, did not show this effect. The addition of small amounts of Na oleate (1 x 10~4 M) to whole cell suspensions greatly stimulated the production of acetaldehyde from threonine (from 2 to 29 /tmoles x 10~4 acetaldehyde produced/min mg (dry wt) cells for Str. lactis subsp. diacetylactis DRC3 and from 10 to 35 /^moles x 10~4 acetaldehyde produced/min mg (dry wt) cells for/Sir. cremoris C13) presumably by increasing the permeability of the bacterial cells (Coles & Lichstein, 1963; Kodicek, 1956). In contrast to the results obtained with whole cell suspensions, with cell-free extracts of Str. lactis subsp. diacetylactis DRC3 a slight inhibition of threonine aldolase activity was obtained with glucose (6% inhibition) or oleate (8% inhibition).
78
G. J. L E E S AND G. R. JAGO
Table 1. Threonine aldolase activities in lactic acid bacteria Organism (A) Group N streptococci
Specific activity!
Streptococcus lactis subsp. diacetylactis DRC1 DRC2
1-29
DRC3 DRC4 18-16
1-56 0-92 0-98 0-37 0-40 0-33 0-59 1-36 0-57 1-28 0-35
Str. lactis
C2 C8F C6 CIO ML3 subsp. maUigenes (MacLeod)
Str. cremoris
HP ML1
0-66 000 0-55 0-59 0-43 0-52 0-40 0-51
E8
Z8 K KH Rl Cl C3 C13
(B) Other lactic acid bacteria Str. faecalis SF1 Str. faecium subsp. durans SD1J Str. thermophilus CSIROJ TW1 ST7
NIZO Lactobacillus bulgariaus%
Leuconostoc cremoris Pediococcus cerevisiae
101
LB1 LB1* LB2* LB3* LB3 LB4*
91404 ATCC 8042*
004 012 0-30 010 0-26 0-33 1-07 0-62 0-69 0-62 0-70 0-70 1-28 001
* Grown in MRS broth. All other organisms grown in tryptone-yeast extract-lactose broth. •(• /tmoles acetaldehyde x 10~2 produced/min mg protein. X Grown at 37 °C. All other organisms grown at 30 °C.
Properties of the threonine aldolase in Str. lactis subsp. diacetylactis DRC3 Under the conditions of assay threonine aldolase activity was linear for at least 60 min. Therefore, an incubation period of 30 min was taken as a measure of the initial reaction velocity. A concentration of L-threonine of 31 mM was found sufficient to saturate the enzyme. The enzyme was found to be specific for the L-isomer of threonine, and the apparent Km value (calculated from a Lineweaver-Burk plot) for the cleavage of this substrate was 3-8 mM. Activity was optimal at pH 7-7 although a relatively high plateau of activity was also present between pH 6-0 and 7-0 (Pig. 3). The stability of the enzyme decreased markedly below pH 5-0 when incubated at 37 °C for 1 h.
Acetaldehyde from threonine (b)
1
79
(c)
16
12
3 2 1f Attenuation: 1 x 16
2 1| 1x16
3
2
1
t
1x8
Retention time, 1 cm = 1 min
Fig. 1. Identification of acetaldehyde, formed from threonine, and its further reduction to othanol in the presence of cell-free extraots of Streptococcus lactis subsp. diacetylactis DRC3. (a) Strain DRC3 + threonine + NADHj, (6) Strain DRC3 + threonine, (c) acetaldehyde(l) + ethanol(2).
Table 2. Effect of temperature and pH of growth on the levels of threonine aldolase in bacterial cell-free extracts Specific activity* after growth at: pH of growth Organism
medium
30 °C
37 CC
Streptococcus lactis subsp. diacetylactis DRC3
NC (final, c. 4-8) 6-3 NC NC NC
1-56
0-78
1-66 111 102 0-40
0-90 107 0-30 003
Str. lactis subsp. diacetylactis DRC3 Lactobacillus bulgaricus LB1 Str. thermophilus CSIRO Escherichia coli K12S
• /(moles acetaldehyde x 10"* produced/min mg protein. All organisms were grown in tryptone-yeastextract broth containing 0-5 % lactose. NC, pH not controlled during growth.
In contrast to the threonine aldolase of Cl. pasteurianum (Dainty, 1967), no requirement for pyridoxal phosphate could be demonstrated for the enzyme in extracts of strain DRC3 dialysed against 0-1 M-Na phosphate buffer, pH 7-0 (to remove pyridoxal phosphate) with or without cysteine. Analogues or derivatives of threonine did not significantly inhibit the total production of carbonyl compounds from threonine. In fact, the production of acetaldehyde from threonine by cell-free extracts was greatly stimulated in the presence of threonyl peptides containing leucine or alanine but not glycine.
80
G. J. L E E S AND G. R. JAGO (a) Solvent front Solvent front
Origin
Origin
Fig. 2. Identification of glyoine as a product of threonine metabolism in Streptococcus laclis subsp. diacetylactis DRC3. (a) 1, Strain DRC3 + water; 2, Strain DRC3 +threonine; 3, Strain DRC3 + glycine + acetaldehyde (156/anole); 4, threonine; 5, glycine + acetaldehyde. Products chromatographed in solvent 1 described under Methods. (6) 1, threonine + glycine; 2, Strain DRC3 + threonine; 3, Strain DRC3 + water; 4, Strain DRC3 + glycine + acetaldehyde (14 /tmole); 5, threonine; 6, allothreonine. Products chromatographed in solvent 2 described under Methods.
Table 3. Stimulation by glucose of the production of acetaldehyde from threonine in bacterial whole cell suspensions (Acetaldehyde + ethanol) • produced from: Organism Streptococcus laclis subsp. diacetylactis DRC3 Str. lactis C2 Str. lactis C8F Str. cremoris HP Str. cremoris Z8 Lactobacillus bulgaricus LB1 Pediococcus cerevisiae ATCC 8042
Glucose 7-02 2-95 0-26
Threonine Threonine + glucose 2-30 4-95 3-56 3-23
11-71 902
5-56
00
018 001
12-95
6-40 25-74 4-93 15-25
o-ot
018
213
1
* /anoles x 10" produced/min mg (dry wt) cells. t Estimated by the semicarbazide method. All other values were estimated by the gas-chromatographic method.
Thiol reagents had little effect on the activity of threonine aldolase. However, thiol compounds containing a free amino group markedly inhibited this activity (Table 4), and as cysteamine inhibited activity a carboxyl group did not appear to be essential for inhibition. Analogues of cysteine which did not contain a thiol group did not
Acetaldehyde from threonine
81
1-2 r-
0-8 -
o E 0.0
04
00 pH
Fig. 3. pH optimum for the production of acetaldehyde from L-threonine by cell-free extracts of Streptococcus lactis subsp. diacetylactis DRC3. The reactions were carried out as described under Methods, using 0' 1 M-N"a phosphate buffers (0) or 0' 1 M-triethanolamine-HCl buffers (O) • The pH values shown refer to the final pHs of the reaction mixtures.
Table 4. Effect of thiol reagents, thiols and analogues of cysteine on the activity of threonine aldolase in cell-free extracts of Str. lactis subsp. diacetylactis DRC3 Compound (A) Thiol reagents —
Iodoacetate .N-ethylmaleimide p-Chloromercuribenzoate (B) Thiola Glutathione (reduced) L-Cysteine D-Cysteine DL-Homocysteine yff-Thio-DL-valine Cysteamine ^-acetyl-L-cysteine y?-thiopropionate a-thioglycerol (C) Analogues of cysteine Cysteic acid L-methionine
Cone, in assay, mM
Belative activity
—
100
0/
/o
11 11 012
98 86 85
25 25 25 25 25 25 25 25 25
88 4 7 3 6 7 85 96 95
25
96
25
94
The reaction mixtures contained DL-threonine (50 mM) as well as the above compounds. The relative activity has been corrected for any interference (less than 3 %) due to carbonyl compounds arising from the thiols or analogues of cysteine.
inhibit. Glutathione or cysteine added to the reaction mixtures did not spontaneously or enzymically reduce the acetaldehyde formed from threonine to ethanol, a possible reason for the non-appearance of acetaldehyde. The threonine aldolase of strain DRC3 was strongly inhibited by cysteine or glycine. Both types of inhibition showed characteristics of allosteric inhibition and were 6
DAR 43
82
G. J. L E E S AND G. R. JAGO 100 r 80 -
10
15
L-cysteine, mM
100 80 ^
60
|
40
ja
£
20 0 10
-20
15
20
25
Glycine, mM
Fig. 4. Inhibition by (a) cysteine and (6) glycine of the threonine aldolase activities in cell-free extracts of Streptoccocus lactis subsp. diacelylactis DRC3: • , pH 6-3; O» pH 7-2; A, pH 7-5; A,pH7-7.
pH-dependent (Fig. 4a, b). Inhibition by glycine was highly specific since alanine, the next higher homologue of glycine, and other analogues such as glyoxylate, ethanolamine, acetamide, taurine and creatine, did not inhibit. However, inhibition by cysteine was not as specific since both cysteine and homocysteine inhibited activity. Alternative pathways for the utilization of threonine did not appear to exist in Str. lactis subsp. diacetylactis DRC3 as NAD- and NADP-dependent threonine dehydrogenases were absent and only low levels of threonine dehydratase (0-07-0-16 x 10~2/tmoles of a-ketobutyrate produced/min mg protein) were present, compared with the relatively high levels of threonine dehydratase (1-56 x 10~2/*moles of aketobutyrate produced/min mg protein) and NAD-dependent threonine dehydrogenase (0-29-0-78 x 10~2 /tmoles of aminoacetone produced/min mg protein) in the control organism, E. coli K12S, grown and assayed under the same conditions. DISCUSSION
The function of threonine aldolase in Group N streptococci may be concerned with the supply of glycine for growth, as Str. cremoris Z8, which did not contain threonine aldolase, has a nutritional requirement for glycine (Reiter & Oram, 1962). The inhibition of threonine aldolase activity by both glycine and cysteine may represent a mechanism by which the biosynthesis of serine from threonine is con-
Acetaldehyde from threonine
83
trolled. It is unknown whether Group N streptococci can form serine from 3-phosphoglyeerie acid. The dependence on pH of the inhibition of threonine aldolase shown by both cysteine and glycine is a property shared by many allosteric enzymes (Cohen, 1965). The rate-limiting step in the cleavage of threonine to acetaldehyde and glycine by whole cells of Group N streptococci would appear to be the transport of threonine into the cell, as glucose stimulated the cleavage of threonine by whole cells, but not by cell-free extracts. Recent work (G. H. Rice & G. R. Jago, unpublished results) has shown that 14C-labelled threonine is actively transported in Str. lactis subsp. diacetylactis DRC3 in the presence of glucose. Threonine aldolase appears to be a major enzyme in the metabolism of threonine by Group N streptococci as a good correlation was observed between the amount of threonine utilized and the amount of acetaldehyde formed by Str. lactis subsp. diacetylactis DRC3, even in the presence of NAD and NADP. Moreover, Str. cremoris Z8, which does not contain a threonine aldolase, did not utilize added threonine. No other enzyme utilizing threonine was found in significant amounts in Str. lactis subsp. diacetylactis DRC3. However, other enzymes involved in the incorporation of threonine into protein and peptidoglycan (Schleifer & Kandler, 1967) may also be present. The low activity of threonine dehydratase may explain, in part, the nutritional requirement of Group N streptococci for isoleucine (Reiter & Oram, 1962), as the formation of a-ketobutyrate is the first step in the bacterial synthesis of isoleucine. This work was supported by grants from the Dairying Research Committee. One of us (G.J.L.) acknowledges the receipt of an Australian Dairy Produce Board Postgraduate Research Studentship and a University of Melbourne Research Scholarship. REFERENCES BILLS, D. D. & DAY, E. A. (1966). Journal of Dairy Science 49, 1473. BOTTAZZI, V. & DELLAGLIO, F. (1967). Journal of Dairy Research 34, 109. COHEN, G. N. (1965). Annual Review of Microbiology 19, 105. COLES, R. S. & LICHSTEIN, H. C. (1963). Archives of Biochemistry and Biophysics 103, 186. DAINTY, R. H. (1967). Biochemical Journal 104, 46P. FRIEDEMANN, T. E. & HAUOEN, G. E. (1943). Journal of Biological Chemistry 147, 415. GREEN, M. L. & ELLIOTT, W. H. (1964). Biochemical Journal 92, 537. HARVEY, R. J. (1960). Journal of Dairy Research 27, 41. KABASEK, M. A. & GKEENBERQ, D. M. (1957). Journal of Biological Chemistry 227, 191. KEENAN, T. W. & BILLS, D. D. (1968). Journal of Dairy Science 51, 1561. KODICEK, E. (1956). In Biochemical Problems of Lipids (2nd international conference, 1953), p. 401. (Eds G. Popjak and E. Le Breton.) London: Butterworths Scientific Publications. KRAUZE, E., KAGAN, Z. S., YAKOVLEVA, V. I. & KRETOVICH, V. L. (1965). Biochemistry (Biokhimiya)
30, 287. LEES, G. J. & JAGO, G. R. (1976). Journal of Dairy Research 43, 63. LENTI, C. & GRILLO, M. A. (1953). Hoppe-Seylers Zeitschrift fur Physiologische Chemie 293, 234. MALKIN, L. I. & GREENBERG, D. M. (1964). Biochimica et Biophysica Acta 85, 117. MAUZERALL, D. & GRANICK, S. (1956). Journal of Biological Chemistry 219, 435. REITER, B. & ORAM, J. D. (1962). Journal of Dairy Research 29, 63. RIARIO-SFORZA, G., PAGANI, R. & MARINELLO, E. (1969). European Journal of Biochemistry 8, 88.
SCHIRCH, L. & GROSS, T. (1968). Journal of Biological Chemistry 243, 5651. SCHLEIFER, K. H. & KANDLER, O. (1967). Archivfiir Mikrobiologie 57, 365. UMBARGER, H. E. & BROWN, B. (1957). Journal of Bacteriology 73, 105. Printed in Oreat Britain 6-2
