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Microbial hydrolysis of diketopiperazines: Different types of diketopiperazine-assimilating bacteria
JOURNALOP FERMENTATION AND BIOENOINEERING Vol. 83, No. 4, 386-388. 1997
Microbial Hydrolysis of Diketopiperazines: Different Types of Diketopiperazine-Assimilating Bacteria HIROSHI KANZAKI,*
SHUJI ODA, AK10 KOBAYASHI,g
AND KAZUYOSHI
KAWAZU
Laboratory of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700, Japan Received 6 December 1996IAccepted 24 January 1997
Arthrobacter sp. 1-3-1 and a coryneform rod bacterium, strain T-1-3-Y, which assimilate cyclo(Gly-Gly) and cyclo(Gly-L-Tyr) at high rates, respectively, were isolated from soil. Both bacteria exhibit dlketopiperazinehydrolyzing activity intracellularly in contrast to the extracellular cyclo(Gly-Gly) hydrolase reported previously. Arthrobacter sp. 1-3-1 shows a much broader substrate specliicity than strain T-1-3-Y, indicating the diversity of diketoplperazine hydrolysis in nature. Fey words: glycine anhydride,
cyclo(Gly-Gly),
cyclo(Gly-L-Tyr),
enrichment
culture]
to belong to the genus Arthrobacter and the coryneform rod bacteria, respectively. The two strains were tested for DKP-hydrolyzing activity by the following method with use of resting cells suspended in 0.85% NaCl or culture supernatants. The reaction was started by addition of 60 ~1 of the cell suspension or the culture supernatant to 40 ~1 of a 6.25 mM substrate solution. Cyclo(Gly-Gly) and cyclo(Gly-L-Tyr) were used as substrates for the reactions catalyzed by resting cells of strains 1-3-1 and T-1-3-Y, respectively. After incubation at 37°C for 1 h, the reaction was stopped by addition of 20 ,~l of 30% trichloroacetic acid solution, and amino acids in the reaction mixture were determined by an amino acid analyzer (JEOL JLC-300). Both strains showed intracellular DKP-hydrolyzing activity in contrast to an extracellular cyclo(Gly-Gly) hydrolase from Bacillus sp. (3), the only DKP-hydrolyzing enzyme reported previously. The resting-cell reactions were dependent on the duration of incubation and the amount of cells present in the reaction mixture. The resting cells of strains l-3-1 and T-l-3-Y hydrolyzed cyclo(Gly-Gly) and cyclo(Gly-L-Tyr), respectively, to produce their component amino acids, not dipeptides. One explanation for this result may be that a DKP-cleaving (dipeptide-producing) reaction was followed by a dipeptide-cleaving reaction which was so rapid that no dipeptides were detected in the reaction mixture. Addition of DKP as a sole carbon source to the culture media was effective for the production of DKP-
DKPs (2,5_diketopiperazines, 2,5-dioxopiperazines, cyclic dipeptides) and their derivatives are widely distributed in nature as secondary metabolites and some of them
have unique bioactivities, e.g., antimicrobial and antitumor activities (1, 2). In spite of the wide distribution of DKPs in nature, little attention has been paid to their metabolism, and therefore, no reports on DKP-hydrolyzing enzymes except for a cyclo(Gly-Gly) hydrolase from Bacillus sp. (3) are available. This situation prompted us to search for and investigate novel DKP-hydrolyzing enzymes. Here we describe isolation of bacteria which assimilate simple DKPs and their characteristics in hydrolysis of DKPs. DKPs were synthesized from the corresponding dipeptides by the method of Kopple and Ghazaarian (4) except for glycine anhydride (cyclo(Gly-Gly)), which was commercially available. Synthesized DKPs were identified by ‘H-NMR, IR and MS spectroscopy, and optical rotation analysis. DKP-assimilating microorganisms were isolated by an enrichment technique as follows. Five different media listed in Table 1, were used. Soil samples (1 g) were suspended in liquid media in 18 mm+tubes and incubated at 28°C for 3 d. Aliquots of the cultures were inoculated into fresh media of the same composition. Several transfers were made until the isolation of single strains on agar plates. Forty-five bacterial strains isolated from soil by enrichment culture with cyclo(Gly-Gly) as a carbon and/or a nitrogen source were inoculated into 5 ml of medium 4 containing cyclo(Gly-Gly) in 18 mm+tubes and cultivated at 28°C for 3 d aerobically. Four strains showing high growth rates (klO> 1.O) were selected and cultivated again in medium 4 containing 1% cyclo(GlyGly). All four of the strains grew very well (Aeo>5.0), while the 260 bacterial strains from our stock showed lower growth rates under the same conditions. Strain l3-1, which showed the highest growth rate, was chosen for the following experiments. By a similar approach but with use of cyclo(Gly-L-Tyr) instead of cyclo(Gly-Gly), strain T-l-3-Y was selected as the strain that assimilated cyclo(Gly-L-Tyr) at the highest rate. From their taxonomic characteristics, strains 1-3-1 and T-l-3-Y were concluded
TABLE 1. Medium compositions in grams per liter Component DKP Glucose
Medium 1
Medium 2
Medium 3
Medium 4
Medium 5
10
10
10 10
1
1
5
(NH4G304
K2HP04
4.8
=wo4
1.5
NaH2P04 MgS04. 7H20 NaCl NaZC03 Yeast extract Polypepton
* Corresponding author. 6 Present address: Department of Biotechnology, Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.
PH
386
2
0.2
0.2 1
0.5
5
15.5
15.5
4.8
8.5 0.2
8.5 0.2
0.5
0.5
0.5 0.5
7.0
7.0
10.0
10
7.0
7.0
NOTES
VOL. 83, 1997 Arth-
Strain T-l 9-Y
387
rp. 1-3-l
Ho (Giy-Ala) Ho (GM-W CyCb (OlY-bu) (gz3, No (Gly+M CyClO (GWN (ZL 80(“rnOV4OlrI)
40
20
0
0
20
40
60 (nmoll40pl)
FIG. 1. Substrate specificity for glycine-containing DKPs. Several glycine-containing DKPs were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The fZ3 and BA bars indicate the amounts of glycine and the counterpart amino acid, respectively, after the reaction. Atfhe
Straln T-l-3-Y
sp. l-3-1
-, 80 (nmoI&d)
40
20
0
0
20
40
(n~oIMOpl~”
FIG. 2. Substrate specificity for tyrosine-containing and related DKPs. Several tyrosine-containing DKPs and cyclo(r.-Phe-r.-Leu) were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The B and lXi bars indicate the amounts of tyrosine (or phenylalanine) and the counterpart amino acid, respectively, after the reaction.
hydrolyzing enzymes in both strains 1-3-1 and T-l-3-Y. Furthermore, during cultivation, the DKP-hydrolyzing activities in the cells rapidly decreased with decreasing concentration of DKP in the medium. These results indicate that the DKP-hydrolyzing activity was induced by the presence of DKP in the culture medium. The optimum temperatures for the reactions catalyzed by resting cells of strains 1-3-1 and T-l-3-Y were 45°C and 40°C, respectively. At temperatures higher than 5O”C, the resting cells of both strains showed little DKPhydrolyzing activity. The resting cells of strain 1-3-1 showed the maximum DKP-hydrolyzing activity at pH 10, while the pH optimum of the DKP-hydrolyzing reaction by the resting T-l-3-Y cells was pH 6.0. The substrate specificities of the resting-cell reactions for DKP, glycine-, tyrosine-, and D-amino acid-contain-
ing DKPs, are shown in Figs. 1, 2 and 3. The differences in the amounts of the component amino acids generated from DKP may be due to the further metabolism of amino acids derived from the DKP substrate. The resting cells of strain 1-3-1 catalyzed the hydrolysis of a variety of glycine-containing DKPs at a considerable rate, while the resting T-l-3-Y cells showed low level of and no activity against cyclo(Gly+Ala) and cyclo(Gly-Gly), respectively (Fig. 1). Interestingly, the resting T-l-3-Y cells could catalyze the hydrolysis of a variety of dipeptides including Gly-Gly at a considerable rate (data not shown), indicating that the cells have a high level of dipeptidase activity. The resting cells of the two strains showed similar substrate specificities for tyrosine-containing DKPs (Fig. 2). rMUnino acid-containing DKPs (cyclo(Gly-DLeu) and cyclo(Gly-o-Ala)) were hardly hydro-
388
KANZAKI ET AL.
J. Strain
T-l-3-Y
FERMENT.BIOENG.,
Arthmbncter sp. 1-3-l
CyClO (Gly-c-Ala)
CyClO (Giy-~-Ala)
FIG. 3. Substrate specificity for n-amino acid-containing and related DKPs. Several p-amino acid-containing and related DKPs were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The IXl and a bars indicate the amounts of glycine and the counterpart amino acid, respectively, after the reaction,
lyzed by the resting T-l-3-Y cells while they were good substrates for the resting 1-3-1 cells (Fig. 3). The resting cells of the DKP-assimilating strains isolated in the present study exhibited a broad spectrum for DKP substrates, while the cyclo(Gly-Gly) hydrolase (3) catalyzed the hydrolysis of only cyclo(Gly-Gly) among a variety of DKPs tested. The results indicate that strains 1-3-1 and T-l-3-Y have novel DKP-hydrolases in the cells. Strain 1-3-1, a cyclo(Gly-Gly)-assimilating strain, showed a much broader specificities for DKPs than did strain T-1-3-Y, a cyclo(Gly-L-Tyr)-assimilating strain. This suggests that use of cyclo(Gly-Gly) without a chiral center facilitated our isolation of bacteria having an enzyme which acts on a variety of DKPs including Damino acid-containing DKP. These results indicate the diversity of DKP hydrolysis in the microbial world.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture to HK. We are grateful to the SC-NMR laboratory of Okayama University and the MS laboratory of the Faculty of Agriculture, Okayama University for assistance in the ‘H-NMR and MS experiments, respectively. REFERENCES 1. Sammes, P. G.: Naturally occurring 2,5-diketopiperazines and related compounds. Fortschr. Chem. Org. Naturst., 32, 51-118 (1975). 2. Prasad, C.: Bioactive cyclic dipeptides. Peptides, 1, 151-164 (1995). 3. Muro, T., Tominaga, Y., and Oknda, S.: Purification and some properties of cyclo(Gly-Gly) hydrolase from a strain of Bacillus sp. no. 1%. Agric. Biol. Chem., 49, 1567-1572 (1985). 4. Kopple, K. D. and Ghazaarian, H. G.: A convenient synthesis of 2,5_diketopiperazines. J. Org. Chem., 33, 862-864 (1968).
Microbial Hydrolysis of Diketopiperazines: Different Types of Diketopiperazine-Assimilating Bacteria HIROSHI KANZAKI,*
SHUJI ODA, AK10 KOBAYASHI,g
AND KAZUYOSHI
KAWAZU
Laboratory of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700, Japan Received 6 December 1996IAccepted 24 January 1997
Arthrobacter sp. 1-3-1 and a coryneform rod bacterium, strain T-1-3-Y, which assimilate cyclo(Gly-Gly) and cyclo(Gly-L-Tyr) at high rates, respectively, were isolated from soil. Both bacteria exhibit dlketopiperazinehydrolyzing activity intracellularly in contrast to the extracellular cyclo(Gly-Gly) hydrolase reported previously. Arthrobacter sp. 1-3-1 shows a much broader substrate specliicity than strain T-1-3-Y, indicating the diversity of diketoplperazine hydrolysis in nature. Fey words: glycine anhydride,
cyclo(Gly-Gly),
cyclo(Gly-L-Tyr),
enrichment
culture]
to belong to the genus Arthrobacter and the coryneform rod bacteria, respectively. The two strains were tested for DKP-hydrolyzing activity by the following method with use of resting cells suspended in 0.85% NaCl or culture supernatants. The reaction was started by addition of 60 ~1 of the cell suspension or the culture supernatant to 40 ~1 of a 6.25 mM substrate solution. Cyclo(Gly-Gly) and cyclo(Gly-L-Tyr) were used as substrates for the reactions catalyzed by resting cells of strains 1-3-1 and T-1-3-Y, respectively. After incubation at 37°C for 1 h, the reaction was stopped by addition of 20 ,~l of 30% trichloroacetic acid solution, and amino acids in the reaction mixture were determined by an amino acid analyzer (JEOL JLC-300). Both strains showed intracellular DKP-hydrolyzing activity in contrast to an extracellular cyclo(Gly-Gly) hydrolase from Bacillus sp. (3), the only DKP-hydrolyzing enzyme reported previously. The resting-cell reactions were dependent on the duration of incubation and the amount of cells present in the reaction mixture. The resting cells of strains l-3-1 and T-l-3-Y hydrolyzed cyclo(Gly-Gly) and cyclo(Gly-L-Tyr), respectively, to produce their component amino acids, not dipeptides. One explanation for this result may be that a DKP-cleaving (dipeptide-producing) reaction was followed by a dipeptide-cleaving reaction which was so rapid that no dipeptides were detected in the reaction mixture. Addition of DKP as a sole carbon source to the culture media was effective for the production of DKP-
DKPs (2,5_diketopiperazines, 2,5-dioxopiperazines, cyclic dipeptides) and their derivatives are widely distributed in nature as secondary metabolites and some of them
have unique bioactivities, e.g., antimicrobial and antitumor activities (1, 2). In spite of the wide distribution of DKPs in nature, little attention has been paid to their metabolism, and therefore, no reports on DKP-hydrolyzing enzymes except for a cyclo(Gly-Gly) hydrolase from Bacillus sp. (3) are available. This situation prompted us to search for and investigate novel DKP-hydrolyzing enzymes. Here we describe isolation of bacteria which assimilate simple DKPs and their characteristics in hydrolysis of DKPs. DKPs were synthesized from the corresponding dipeptides by the method of Kopple and Ghazaarian (4) except for glycine anhydride (cyclo(Gly-Gly)), which was commercially available. Synthesized DKPs were identified by ‘H-NMR, IR and MS spectroscopy, and optical rotation analysis. DKP-assimilating microorganisms were isolated by an enrichment technique as follows. Five different media listed in Table 1, were used. Soil samples (1 g) were suspended in liquid media in 18 mm+tubes and incubated at 28°C for 3 d. Aliquots of the cultures were inoculated into fresh media of the same composition. Several transfers were made until the isolation of single strains on agar plates. Forty-five bacterial strains isolated from soil by enrichment culture with cyclo(Gly-Gly) as a carbon and/or a nitrogen source were inoculated into 5 ml of medium 4 containing cyclo(Gly-Gly) in 18 mm+tubes and cultivated at 28°C for 3 d aerobically. Four strains showing high growth rates (klO> 1.O) were selected and cultivated again in medium 4 containing 1% cyclo(GlyGly). All four of the strains grew very well (Aeo>5.0), while the 260 bacterial strains from our stock showed lower growth rates under the same conditions. Strain l3-1, which showed the highest growth rate, was chosen for the following experiments. By a similar approach but with use of cyclo(Gly-L-Tyr) instead of cyclo(Gly-Gly), strain T-l-3-Y was selected as the strain that assimilated cyclo(Gly-L-Tyr) at the highest rate. From their taxonomic characteristics, strains 1-3-1 and T-l-3-Y were concluded
TABLE 1. Medium compositions in grams per liter Component DKP Glucose
Medium 1
Medium 2
Medium 3
Medium 4
Medium 5
10
10
10 10
1
1
5
(NH4G304
K2HP04
4.8
=wo4
1.5
NaH2P04 MgS04. 7H20 NaCl NaZC03 Yeast extract Polypepton
* Corresponding author. 6 Present address: Department of Biotechnology, Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.
PH
386
2
0.2
0.2 1
0.5
5
15.5
15.5
4.8
8.5 0.2
8.5 0.2
0.5
0.5
0.5 0.5
7.0
7.0
10.0
10
7.0
7.0
NOTES
VOL. 83, 1997 Arth-
Strain T-l 9-Y
387
rp. 1-3-l
Ho (Giy-Ala) Ho (GM-W CyCb (OlY-bu) (gz3, No (Gly+M CyClO (GWN (ZL 80(“rnOV4OlrI)
40
20
0
0
20
40
60 (nmoll40pl)
FIG. 1. Substrate specificity for glycine-containing DKPs. Several glycine-containing DKPs were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The fZ3 and BA bars indicate the amounts of glycine and the counterpart amino acid, respectively, after the reaction. Atfhe
Straln T-l-3-Y
sp. l-3-1
-, 80 (nmoI&d)
40
20
0
0
20
40
(n~oIMOpl~”
FIG. 2. Substrate specificity for tyrosine-containing and related DKPs. Several tyrosine-containing DKPs and cyclo(r.-Phe-r.-Leu) were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The B and lXi bars indicate the amounts of tyrosine (or phenylalanine) and the counterpart amino acid, respectively, after the reaction.
hydrolyzing enzymes in both strains 1-3-1 and T-l-3-Y. Furthermore, during cultivation, the DKP-hydrolyzing activities in the cells rapidly decreased with decreasing concentration of DKP in the medium. These results indicate that the DKP-hydrolyzing activity was induced by the presence of DKP in the culture medium. The optimum temperatures for the reactions catalyzed by resting cells of strains 1-3-1 and T-l-3-Y were 45°C and 40°C, respectively. At temperatures higher than 5O”C, the resting cells of both strains showed little DKPhydrolyzing activity. The resting cells of strain 1-3-1 showed the maximum DKP-hydrolyzing activity at pH 10, while the pH optimum of the DKP-hydrolyzing reaction by the resting T-l-3-Y cells was pH 6.0. The substrate specificities of the resting-cell reactions for DKP, glycine-, tyrosine-, and D-amino acid-contain-
ing DKPs, are shown in Figs. 1, 2 and 3. The differences in the amounts of the component amino acids generated from DKP may be due to the further metabolism of amino acids derived from the DKP substrate. The resting cells of strain 1-3-1 catalyzed the hydrolysis of a variety of glycine-containing DKPs at a considerable rate, while the resting T-l-3-Y cells showed low level of and no activity against cyclo(Gly+Ala) and cyclo(Gly-Gly), respectively (Fig. 1). Interestingly, the resting T-l-3-Y cells could catalyze the hydrolysis of a variety of dipeptides including Gly-Gly at a considerable rate (data not shown), indicating that the cells have a high level of dipeptidase activity. The resting cells of the two strains showed similar substrate specificities for tyrosine-containing DKPs (Fig. 2). rMUnino acid-containing DKPs (cyclo(Gly-DLeu) and cyclo(Gly-o-Ala)) were hardly hydro-
388
KANZAKI ET AL.
J. Strain
T-l-3-Y
FERMENT.BIOENG.,
Arthmbncter sp. 1-3-l
CyClO (Gly-c-Ala)
CyClO (Giy-~-Ala)
FIG. 3. Substrate specificity for n-amino acid-containing and related DKPs. Several p-amino acid-containing and related DKPs were tested as substrates for hydrolysis by the resting cells of strains 1-3-1 and T-l-3-Y. The IXl and a bars indicate the amounts of glycine and the counterpart amino acid, respectively, after the reaction,
lyzed by the resting T-l-3-Y cells while they were good substrates for the resting 1-3-1 cells (Fig. 3). The resting cells of the DKP-assimilating strains isolated in the present study exhibited a broad spectrum for DKP substrates, while the cyclo(Gly-Gly) hydrolase (3) catalyzed the hydrolysis of only cyclo(Gly-Gly) among a variety of DKPs tested. The results indicate that strains 1-3-1 and T-l-3-Y have novel DKP-hydrolases in the cells. Strain 1-3-1, a cyclo(Gly-Gly)-assimilating strain, showed a much broader specificities for DKPs than did strain T-1-3-Y, a cyclo(Gly-L-Tyr)-assimilating strain. This suggests that use of cyclo(Gly-Gly) without a chiral center facilitated our isolation of bacteria having an enzyme which acts on a variety of DKPs including Damino acid-containing DKP. These results indicate the diversity of DKP hydrolysis in the microbial world.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture to HK. We are grateful to the SC-NMR laboratory of Okayama University and the MS laboratory of the Faculty of Agriculture, Okayama University for assistance in the ‘H-NMR and MS experiments, respectively. REFERENCES 1. Sammes, P. G.: Naturally occurring 2,5-diketopiperazines and related compounds. Fortschr. Chem. Org. Naturst., 32, 51-118 (1975). 2. Prasad, C.: Bioactive cyclic dipeptides. Peptides, 1, 151-164 (1995). 3. Muro, T., Tominaga, Y., and Oknda, S.: Purification and some properties of cyclo(Gly-Gly) hydrolase from a strain of Bacillus sp. no. 1%. Agric. Biol. Chem., 49, 1567-1572 (1985). 4. Kopple, K. D. and Ghazaarian, H. G.: A convenient synthesis of 2,5_diketopiperazines. J. Org. Chem., 33, 862-864 (1968).