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Molecular cloning and characterization of l -methionine γ-lyase from Streptomyces avermitilis
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e4, 2015 www.elsevier.com/locate/jbiosc
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Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis Daizou Kudou, Eri Yasuda, Yoshiyuki Hirai, Takashi Tamura, and Kenji Inagaki* Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama-shi, Okayama 700-8530, Japan Received 20 August 2014; accepted 28 February 2015 Available online xxx
A pyridoxal 50 -phosphate-dependent methionine g-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to a-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pyridoxal 50 -phosphate; L-Methionine g-lyase; Streptomyces avermitilis; Elimination activity; Replacement activity; Substrate specificity; TLC analysis; Kinetic analysis; Phylogenetic analysis] 0 L-Methionine g-lyase (MGL, EC 4.4.1.11) is a pyridoxal 5 -phosphate (PLP)-dependent enzyme, catalyzing the a,g-elimination of Lmethionine to produce equimolar amounts of methanethiol, ammonia, and a-ketobutyrate. The enzyme also catalyzes the belimination reaction of L-cysteine and S-substituted L-cysteines as well as the g- and b-replacement reactions of L-methionine, Lcysteine and their analogs. MGL has been isolated from Pseudomonas putida, from some other bacteria, from the primitive protozoa Trichomonas vaginalis and Entamoeba histolytica, and from the plant Arabidopsis thaliana (1e9). The avermectins are oleandrose disaccharide derivatives of 16 membered pentacyclic lactones which are produced by S. avermitilis. The S-methyl of methionine is extensively and equally incorporated into the three methoxyl groups of the avermectins (10). The methyltransferases, which transfer a methyl group from a methyl donor such as S-adenosyl-L-methionine to secondary metabolites produced in Streptomyces, play an important role in the modification of antibiotics (11). However, little has been reported on methionine metabolism, especially on methionine degradation, in Streptomyces. Therefore, we focused on the methionine degradation enzyme, methionine g-lyase, in S. avermitilis. The complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined (12). In silico we found the putative candidate gene (sav7062) coding for L-methionine glyase on the chromosome of the Streptomycete strain to compare it with well-studied version of this enzyme from P. putida. An
* Corresponding author. Tel./fax: þ81 86 251 8299. E-mail addresses: [email protected] (D. Kudou), ag422114@ s.okayama-u.ac.jp (E. Yasuda), [email protected] (Y. Hirai), [email protected] (T. Tamura), [email protected] (K. Inagaki).
open reading frame of 1239 bp was identified, corresponding to 413 amino acid residues with a molecular mass of 42,994 Da. Our group has set out to acquire high resolution structural data for MGL from P. putida (MGL_Pp) (13). The homotetrameric MGLs from various organisms are closely related in their overall structures as well as in the arrangement of several active-site residues. Tyr114 and Cys116 (the number is derived from MGL_Pp) are conserved in heterologous MGLs and it is thought that these residues are important in forming the catalytic pocket of this enzyme (14,15). In the amino acid sequence of the sav7062 gene from S. avermitilis, the fact that these residues are highly conserved in silico raises the possibility that the gene codes for Lmethionine g-lyase (Fig. 1). The deduced amino acid sequence was used to search for similar sequences in protein databases with the Basic Local Alignment Search Tool (ClustalW, URL: http://align. genome.jp/). This amino acid sequence shared 31e39% similarity with the sequences of MGL genes of P. putida (36%), Brevibacterium linens (32%), Citrobacter freundii (39%), E. histolytica (36%), Treponema denticola (38%), T. vaginalis (39%) and A. thaliana (31%). The important residues of MGL_Pp are stringently conserved in the SAV7062 of S. avermitilis: Lys221 and Tyr131 are corresponding to the essential residues of Lys211 and Tyr114 of MGL_Pp, Cys133 is corresponding to Cys116 of MGL_Pp, which is important residue of the hydrogen bond network in the active site, and Tyr75 and Arg77 are corresponding to Tyr59 and Arg61 of MGL_Pp, which are residues of the binding of the PLP phosphate group and potential candidates for substrate- and cofactor-binding residues (13). Here, we have described the construction of the high expression system of the sav7062 gene from S. avermitilis in E. coli, as well as the purification, and characterization of its gene product, L-methionine g-lyase, to clarify its enzymological and structural properties.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
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FIG. 1. Alignment of amino acid in MGL from P. putida and S. avermitilis. Arrow points to putative active site residues. Conserved residues are shown with asterisks, and homologs with dots and colons.
A full-length DNA coding for SAV7062 (MGL_Sa) was obtained by PCR amplification of S. avermitilis genome DNA. Primers SaMGL1 (50 -AAAACCATGGACGACGGCCGGGGCGCCG-30 ) that overlap the initiation Met codon (italic) and SaMGL2 (50 -TTTGCGGCCGCG GCCCGCTCCGGGAGCCGG-30 ), containing Nco I site and Not I site (underlined), respectively, were used for the cloning of MGL_Sa into the pET23d(þ) expression vector (Novagen). The resulting plasmid, pSM7, encodes a fusion between the complete ORF of SAV7062 and a C-terminal hexa-histidine tag. As there are four C-terminal regions on the enzyme surface in most MGLs, it was speculated that this tag had no effect on enzyme activity. The pSM7 construct was introduced into E. coli BL21(DE3) pLysS (Stratagene). E. coli BL21 cells harboring pSM7 were grown in a 5 mL LB medium containing ampicillin (100 mg/mL, pH 7.0) at 37 C for 15 h with vigorous shaking. The culture broth was transferred to a 400 mL cultivation medium (1.2% tryptone, 2.4% yeast extract, 2.0% glycerol, 1.25% K2HPO4, 0.23% KH2PO4, 0.05% polypropylene glycol no. 2000, 100mg/mL ampicillin, and 34 mg/mL chloramphenicol, pH 7.0) and incubated at 28 C for 2 h. Isopropylthio-b-D-galactoside was added (0.2 mM), and the cells were grown further for 20 h at 28 C. Cells from the culture were collected by centrifugation at 6000 g for 30 min, suspended in buffer A (50 mM potassium phosphate buffer (KPB), pH 8.0, with 0.01 mM PLP and 0.3 M KCl) containing 10 mM of imidazole, and then disrupted by sonication. The soluble protein extract was separated from cell debris by centrifugation at 20,000 g for 30 min and applied onto a nickelnitrilotriacetic acid-agarose column (Ni-NTA, QIAGEN) previously equilibrated with buffer A. After successive washes with buffer A supplemented with 10, 20, and 50 mM of imidazole, the recombinant protein was eluted with buffer A containing 100 mM of imidazole. Fractions containing MGL_Sa were pooled and concentrated by centrifugation (Amicon Ultra-15, 30 kD cut-off; Millipore). The enzyme was purified to homogeneity, resulting in a 278-fold purification of a 31.6% yield. The production of a-keto acids, aketobutyrate and pyruvate, was measured using the MBTH method, according to the method of Takakura et al. (16). Enzymes were added to each substrate solution, 100 mM KPB (pH 8.0) with 10 mM PLP containing 25 mM L-methionine, 25 mM L-homocysteine and 5 mM L-cysteine, respectively, and reacted for 10 min at 37 C. The reaction were stopped by adding 50% trichroloacetic acid (TCA) and reacted with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). These elimination reactions proceeded linearly for 10 min (data not shown). One unit of activity is defined as the amount of MGL that catalyzed the formation of 1 mmol of a-ketobutyrate and pyruvate per minute at 37 C, respectively.
To confirm the MGL activity of SAV7062, we tried to detect the reductants for methionine degradation and analyze the kinetic parameters toward three substrates, L-methionine, L-homocysteine, and L-cysteine, using the purified enzyme, respectively. First, the purified enzyme was dialyzed with 20 mM KPB (pH 7.2) containing 1 mM ethylenediamine tetraacetate (EDTA), 0.1 mM PLP and 0.01% dithiothreitol. a-Ketobutyrate was identified by the thin-layer chromatography (TLC) analysis (Fig. S1). The eluent was phenol-water (4:1, w/v) in the elimination reaction for Lmethionine. The thin-layer plate was sprayed with 34 mg/mL 2,4-dinitrophenylhydrazine solution (DNP) with 17% concentrated sulfuric acid and 57% ethanol and heated. This result showed that one of the reductant for methionine breakdown by SAV7062 is a-ketobutyrate. Secondly, the purified enzyme was dialyzed with 20 mM KPB (pH 7.2) containing 1 mM EDTA, 0.1 mM PLP and the methanethiol was detected using 5,50 dithiobis-2-nitrobenzoic acid (DTNB). Thirty microliter of the reaction mixture of methionine degradation, which was the same mixture at the MBTH method as mentioned above, was mixed to 1.5 mL of 200 mM sodium phosphate buffer (pH 7.0) and 6 mL of 4 mg/mL DTNB and filled up to 3 mL with water. The mixture was left at room temperature for 5 min and measured the optical density at 412 nm. For 10 min reaction, 0.05 mM methanethiol was produced, which was the same concentration of a-ketobutyrate. Consequently, the SAV7062 enzyme has an activity of methionine g-lyase. Next, the Km values for each substrate were similar to those of MGL_Pp; however, the kcat values toward Lmethionine and L-homocysteine were lower than those of MGL_Pp by 0.11 fold and 0.04 fold, respectively (Table 1). The activity toward L-cysteine was little, whereas the substrate specificity was higher than that of MGL_Pp (15). The phylogenetic tree of MGLs and g-family enzymes was made as shown in Fig. 2. The g-family enzymes of PLP-dependent enzyme group have a,g-elimination, g-replacement, a,b-elimination, or b-replacement reaction activities and are commonly suicide-inactivated with L-propargylglycine (17). Interestingly, the
TABLE 1. The kinetic parameters of MGL_Sa.
L-Methionine L-Homocysteine L-Cysteine
Km (mM)
kcat (s1)
1.60 1.78 0.20
2.90 1.67 0.03
kcat/Km (s1mM1) 1.81 0.94 0.15
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
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FIG. 2. Phylogenic tree of g-family enzymes. Figure was drawn using ClustalW. OAHS_Tt, O-acetyl-L-homoserine sulfhydrylase from Thermus thermophilus; OAHS_Pp, O-acetyl-Lhomoserine sulfhydrylase from Pseudomonas putida; OSHS_Pp, O-succinyl-L-homoserine sulfhydrylase from P. putida; CBL_Sc, cystathionine b-lyase from Saccharomyces cerevisiae; CBL_Ll, cystathionine b-lyase from Lactococcus lactis; CBL_At, cystathionine b-lyase from Arabidopsis thaliana; CBL_Ec, cystathionine b-lyase from Escherichia coli; CGS_Nt, cystathionine g-synthase from Nicotiana tabacum; CGS_At, cystathionine g-synthase from A. thaliana; CGS_Ec, cystathionine g-synthase from E. coli; MGL_Bl, L-methionine g-lyase from Brevibacterium linens; MGL_Cf, L-methionine g-lyase from Citrobacter freundii; MGL_Eh, L-methionine g-lyase from Entamoeba histolytica; MGL_Pp, L-methionine g-lyase from P. putida; MGL_Td, L-methionine g-lyase from Treponema denticola; MGL_Tv, L-methionine g-lyase from Trichomonas vaginalis; MGL_At, L-methionine g-lyase from A. thaliana; MGL_Sa, L-methionine g-lyase from S. avermitilis.
MGL_Sa from S. avermitilis formed a cluster with the sulfhydrylase group and was closer to the cystathionine g-synthase and cystathionine b-lyase than MGL from P. putida. Then, substrate specificity for some elimination substrates was investigated (Table S1). The g-elimination activities toward g-elimination substrates, DL-homocysteine and O-succinyl-L-homoserine, were lower than that of L-methionine. The data for the b-elimination activities toward cystathionine, L-cysteine, S-methyl-L-cysteine, Sethyl-L-cysteine, O-phospho-L-serine, O-acetyl-L-serine and Oacetyl-L-serine showed a similar tendency like as MGL_Pp (15). Furthermore, it was found that MGL_Sa has lower g-replacement activities than those of MGL_Pp by TLC analysis (Fig. S2). Cystathionine synthesis reaction of MGL_Sa and MGL_Pp was carried out under the condition as follows: 20 mL substrate solution in 100 mM KPB (pH 8.0) with 100 mM O-succinyl-L-homoserine, 100 mM L-cysteine and 10 mM PLP was prepared, 5 mL enzyme solutions were added, respectively, and these were reactioned at 37 C for 1 h. These were stopped by adding 2.5 mL of 50% TCA. Cystathionine synthesis reaction of cystathionine g-synthase from Sulfolobus tokodaii (CGS_St) was carried out under the condition as follows: 20 mL substrate solution in 100 mM TriseHCl (pH 9.0) with 100 mM O-succinyl-L-homoserine, 100 mM L-cysteine and 10 mM PLP was prepared, 5 mL enzyme solution was added and it was reactioned at 70 C for 1 h. It was stopped by adding 2.5 mL of 50% TCA. S-(b-hydroxyethyl) homocysteine synthesis reaction of MGL_Sa and MGL_Pp was carried out under the same conditions in cystathionine synthesis, replacing L-cysteine into 160 mM 2mercaptoethanol (18). S-(b-hydroxyethyl) homocysteine synthesis reaction of CGS_St was also carried out under the same
conditions in cystathionine synthesis, replacing L-cysteine into 160 mM 2-mercaptoethanol. These data showed that SAV7062 has no replacement activity for cystathionine and S-(b-hydroxyethyl) homocysteine synthesis, indicating SAV7062 is L-methionine glyase and interestingly has higher reaction specificity than MGL_Pp. For the phylogenetic analysis it was found that this protein was clearly separated from heterologous MGLs in the early stages of evolution and was nearly similar to plant MGLs (Fig. 2). This report was mainly about MGLs from Streptomycete and was not only concerned with phylogenic trees but also MGL evolution. In this study the enzyme activity of SAV7062 as a MGL involved in S. avermitilis sulfur metabolism was clarified partially, however there are little data to speculate the physiological roles of this enzyme in Streptomycete. We wish to keep in analyzing the sulfur metabolism in Streptomycete to clarify the mechanism the secondary metabolism. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.02.019 References 1. Nakayama, T., Esaki, N., Sugie, K., Beresov, T. T., Tanaka, H., and Soda, K.: Purification of bacterial L-methionine g-lyase, Anal. Biochem., 138, 421e424 (1984). 2. Nakayama, T., Esaki, N., Lee, W. J., Tanaka, I., Tanaka, H., and Soda, K.: Purification and properties of L-methionine g-lyase from Aeromonas sp. Agric. Biol. Chem., 48, 2367e2369 (1984). 3. Manukhov, I. V., Mamaeva, D. V., Rastorguev, S. M., Faleev, N. G., Morozova, E. A., Demidkina, T. V., and Zavilgelsky, G. B.: A gene encoding Lmethionine g-lyase is present in Enterobacteriaceae family genomes:
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
4
4.
5.
6.
7. 8.
9.
10.
11.
KUDOU ET AL. identification and characterization of Citrobacter freundii L-methionine g-lyase, J. Bacteriol., 187, 3889e3893 (2005). Amarita, F., Yvon, M., Nardi, M., Chambellon, E., Delettre, J., and Bonnarme, P.: Identification and functional analysis of the gene encoding methionine g-lyase in Brevibacterium linens, Appl. Environ. Microbiol., 70, 7348e7354 (2004). Yoshimura, M., Nakano, Y., Yamashita, Y., Oho, T., Saito, T., and Koga, T.: Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis, Infect. Immun., 68, 6912e6916 (2000). Fukamachi, H., Nakano, Y., Okano, S., Shibata, Y., Abiko, Y., and Yamashita, Y.: High production of methyl mercaptan by L-methionine-a-deamino-g-mercaptomethane lyase from Treponema denticola, Biochem. Biophys. Res. Commun., 331, 127e131 (2005). Lockwood, B. C. and Coombs, G. H.: Purification and characterization of methionine g-lyase from Trichomonas vaginalis, Biochem. J., 279, 675e682 (1991). Tokoro, M., Asai, T., Kobayashi, S., Takeuchi, T., and Nozaki, T.: Identification and characterization of two isoenzymes of methionine g-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways, J. Biol. Chem., 278, 42717e42727 (2003). Rébeillé, F., Jabrin, S., Bligny, R., Loizeau, K., Gambonnet, B., Van Wilder, V., Douce, R., and Ravanel, S.: Methionine catabolism in Arabidopsis cells is initiated by a g-cleavage process and leads to S-methylcysteine and isoleucine syntheses, Proc. Natl. Acad. Sci. USA, 103, 15687e15692 (2006). Marvin, D. S., Delia, V., and Otto, H.: Biosynthesis of the avermectins by Streptomyces avermitilis, incorporation of labeled precursors, J. Antibiot. (Tokyo), 39, 541e549 (1986). Yoon, Y., Younghee, P., Yong, S. Y., Youngshim, L., Geunhyeong, J., Jun, C. P., Joong, H. A., and Yoongho, L.: Characterization of an O-methyltransferase
J. BIOSCI. BIOENG.,
12.
13.
14.
15.
16.
17.
18.
from Streptomyces avermitilis MA-4680, J. Microbiol. Biotechnol., 20, 1359e1366 (2010). Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M., and Omura, S.: Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis, Nat. Biotechnol., 21, 526e531 (2003). Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takakura, T., Yoshioka, T., Yagi, S., Hoffman, R. M., Takimoto, A., Esaki, N., and Inagaki, K.: Structure of the antitumour enzyme L-methionine g-lyase from Pseudomonas putida at 1.8 Å resolution, J. Biochem., 141, 535e544 (2007). Inoue, H., Inagaki, K., Adachi, N., Tamura, T., Esaki, N., Soda, K., and Tanaka, H.: Role of tyrosine 114 of L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 64, 2336e2343 (2000). Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Esaki, N., and Inagaki, K.: The role of cysteine 116 in the active site of the antitumor enzyme L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 72, 1722e1730 (2008). Takakura, T., Mitsushima, K., Yagi, S., Inagaki, K., Tanaka, H., Esaki, N., Soda, K., and Takimoto, A.: Assay method for antitumor L-methionine g-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine, Anal. Biochem., 327, 233e240 (2004). Johnston, M., Jankowski, D., Marcotte, P., Tanaka, H., Esaki, N., Soda, K., and Walsh, C.: Suicide inactivation of bacterial cystathionine g-synthase and methionine g-lyase during processing of L-propargylglycine, Biochemistry, 18, 4690e4701 (1979). Harada, S., and Inagaki, K.: The role of amino acids residues in the active site of L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 76, 1275e1284 (2012).
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
NOTE
Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis Daizou Kudou, Eri Yasuda, Yoshiyuki Hirai, Takashi Tamura, and Kenji Inagaki* Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama-shi, Okayama 700-8530, Japan Received 20 August 2014; accepted 28 February 2015 Available online xxx
A pyridoxal 50 -phosphate-dependent methionine g-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to a-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pyridoxal 50 -phosphate; L-Methionine g-lyase; Streptomyces avermitilis; Elimination activity; Replacement activity; Substrate specificity; TLC analysis; Kinetic analysis; Phylogenetic analysis] 0 L-Methionine g-lyase (MGL, EC 4.4.1.11) is a pyridoxal 5 -phosphate (PLP)-dependent enzyme, catalyzing the a,g-elimination of Lmethionine to produce equimolar amounts of methanethiol, ammonia, and a-ketobutyrate. The enzyme also catalyzes the belimination reaction of L-cysteine and S-substituted L-cysteines as well as the g- and b-replacement reactions of L-methionine, Lcysteine and their analogs. MGL has been isolated from Pseudomonas putida, from some other bacteria, from the primitive protozoa Trichomonas vaginalis and Entamoeba histolytica, and from the plant Arabidopsis thaliana (1e9). The avermectins are oleandrose disaccharide derivatives of 16 membered pentacyclic lactones which are produced by S. avermitilis. The S-methyl of methionine is extensively and equally incorporated into the three methoxyl groups of the avermectins (10). The methyltransferases, which transfer a methyl group from a methyl donor such as S-adenosyl-L-methionine to secondary metabolites produced in Streptomyces, play an important role in the modification of antibiotics (11). However, little has been reported on methionine metabolism, especially on methionine degradation, in Streptomyces. Therefore, we focused on the methionine degradation enzyme, methionine g-lyase, in S. avermitilis. The complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined (12). In silico we found the putative candidate gene (sav7062) coding for L-methionine glyase on the chromosome of the Streptomycete strain to compare it with well-studied version of this enzyme from P. putida. An
* Corresponding author. Tel./fax: þ81 86 251 8299. E-mail addresses: [email protected] (D. Kudou), ag422114@ s.okayama-u.ac.jp (E. Yasuda), [email protected] (Y. Hirai), [email protected] (T. Tamura), [email protected] (K. Inagaki).
open reading frame of 1239 bp was identified, corresponding to 413 amino acid residues with a molecular mass of 42,994 Da. Our group has set out to acquire high resolution structural data for MGL from P. putida (MGL_Pp) (13). The homotetrameric MGLs from various organisms are closely related in their overall structures as well as in the arrangement of several active-site residues. Tyr114 and Cys116 (the number is derived from MGL_Pp) are conserved in heterologous MGLs and it is thought that these residues are important in forming the catalytic pocket of this enzyme (14,15). In the amino acid sequence of the sav7062 gene from S. avermitilis, the fact that these residues are highly conserved in silico raises the possibility that the gene codes for Lmethionine g-lyase (Fig. 1). The deduced amino acid sequence was used to search for similar sequences in protein databases with the Basic Local Alignment Search Tool (ClustalW, URL: http://align. genome.jp/). This amino acid sequence shared 31e39% similarity with the sequences of MGL genes of P. putida (36%), Brevibacterium linens (32%), Citrobacter freundii (39%), E. histolytica (36%), Treponema denticola (38%), T. vaginalis (39%) and A. thaliana (31%). The important residues of MGL_Pp are stringently conserved in the SAV7062 of S. avermitilis: Lys221 and Tyr131 are corresponding to the essential residues of Lys211 and Tyr114 of MGL_Pp, Cys133 is corresponding to Cys116 of MGL_Pp, which is important residue of the hydrogen bond network in the active site, and Tyr75 and Arg77 are corresponding to Tyr59 and Arg61 of MGL_Pp, which are residues of the binding of the PLP phosphate group and potential candidates for substrate- and cofactor-binding residues (13). Here, we have described the construction of the high expression system of the sav7062 gene from S. avermitilis in E. coli, as well as the purification, and characterization of its gene product, L-methionine g-lyase, to clarify its enzymological and structural properties.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
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FIG. 1. Alignment of amino acid in MGL from P. putida and S. avermitilis. Arrow points to putative active site residues. Conserved residues are shown with asterisks, and homologs with dots and colons.
A full-length DNA coding for SAV7062 (MGL_Sa) was obtained by PCR amplification of S. avermitilis genome DNA. Primers SaMGL1 (50 -AAAACCATGGACGACGGCCGGGGCGCCG-30 ) that overlap the initiation Met codon (italic) and SaMGL2 (50 -TTTGCGGCCGCG GCCCGCTCCGGGAGCCGG-30 ), containing Nco I site and Not I site (underlined), respectively, were used for the cloning of MGL_Sa into the pET23d(þ) expression vector (Novagen). The resulting plasmid, pSM7, encodes a fusion between the complete ORF of SAV7062 and a C-terminal hexa-histidine tag. As there are four C-terminal regions on the enzyme surface in most MGLs, it was speculated that this tag had no effect on enzyme activity. The pSM7 construct was introduced into E. coli BL21(DE3) pLysS (Stratagene). E. coli BL21 cells harboring pSM7 were grown in a 5 mL LB medium containing ampicillin (100 mg/mL, pH 7.0) at 37 C for 15 h with vigorous shaking. The culture broth was transferred to a 400 mL cultivation medium (1.2% tryptone, 2.4% yeast extract, 2.0% glycerol, 1.25% K2HPO4, 0.23% KH2PO4, 0.05% polypropylene glycol no. 2000, 100mg/mL ampicillin, and 34 mg/mL chloramphenicol, pH 7.0) and incubated at 28 C for 2 h. Isopropylthio-b-D-galactoside was added (0.2 mM), and the cells were grown further for 20 h at 28 C. Cells from the culture were collected by centrifugation at 6000 g for 30 min, suspended in buffer A (50 mM potassium phosphate buffer (KPB), pH 8.0, with 0.01 mM PLP and 0.3 M KCl) containing 10 mM of imidazole, and then disrupted by sonication. The soluble protein extract was separated from cell debris by centrifugation at 20,000 g for 30 min and applied onto a nickelnitrilotriacetic acid-agarose column (Ni-NTA, QIAGEN) previously equilibrated with buffer A. After successive washes with buffer A supplemented with 10, 20, and 50 mM of imidazole, the recombinant protein was eluted with buffer A containing 100 mM of imidazole. Fractions containing MGL_Sa were pooled and concentrated by centrifugation (Amicon Ultra-15, 30 kD cut-off; Millipore). The enzyme was purified to homogeneity, resulting in a 278-fold purification of a 31.6% yield. The production of a-keto acids, aketobutyrate and pyruvate, was measured using the MBTH method, according to the method of Takakura et al. (16). Enzymes were added to each substrate solution, 100 mM KPB (pH 8.0) with 10 mM PLP containing 25 mM L-methionine, 25 mM L-homocysteine and 5 mM L-cysteine, respectively, and reacted for 10 min at 37 C. The reaction were stopped by adding 50% trichroloacetic acid (TCA) and reacted with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). These elimination reactions proceeded linearly for 10 min (data not shown). One unit of activity is defined as the amount of MGL that catalyzed the formation of 1 mmol of a-ketobutyrate and pyruvate per minute at 37 C, respectively.
To confirm the MGL activity of SAV7062, we tried to detect the reductants for methionine degradation and analyze the kinetic parameters toward three substrates, L-methionine, L-homocysteine, and L-cysteine, using the purified enzyme, respectively. First, the purified enzyme was dialyzed with 20 mM KPB (pH 7.2) containing 1 mM ethylenediamine tetraacetate (EDTA), 0.1 mM PLP and 0.01% dithiothreitol. a-Ketobutyrate was identified by the thin-layer chromatography (TLC) analysis (Fig. S1). The eluent was phenol-water (4:1, w/v) in the elimination reaction for Lmethionine. The thin-layer plate was sprayed with 34 mg/mL 2,4-dinitrophenylhydrazine solution (DNP) with 17% concentrated sulfuric acid and 57% ethanol and heated. This result showed that one of the reductant for methionine breakdown by SAV7062 is a-ketobutyrate. Secondly, the purified enzyme was dialyzed with 20 mM KPB (pH 7.2) containing 1 mM EDTA, 0.1 mM PLP and the methanethiol was detected using 5,50 dithiobis-2-nitrobenzoic acid (DTNB). Thirty microliter of the reaction mixture of methionine degradation, which was the same mixture at the MBTH method as mentioned above, was mixed to 1.5 mL of 200 mM sodium phosphate buffer (pH 7.0) and 6 mL of 4 mg/mL DTNB and filled up to 3 mL with water. The mixture was left at room temperature for 5 min and measured the optical density at 412 nm. For 10 min reaction, 0.05 mM methanethiol was produced, which was the same concentration of a-ketobutyrate. Consequently, the SAV7062 enzyme has an activity of methionine g-lyase. Next, the Km values for each substrate were similar to those of MGL_Pp; however, the kcat values toward Lmethionine and L-homocysteine were lower than those of MGL_Pp by 0.11 fold and 0.04 fold, respectively (Table 1). The activity toward L-cysteine was little, whereas the substrate specificity was higher than that of MGL_Pp (15). The phylogenetic tree of MGLs and g-family enzymes was made as shown in Fig. 2. The g-family enzymes of PLP-dependent enzyme group have a,g-elimination, g-replacement, a,b-elimination, or b-replacement reaction activities and are commonly suicide-inactivated with L-propargylglycine (17). Interestingly, the
TABLE 1. The kinetic parameters of MGL_Sa.
L-Methionine L-Homocysteine L-Cysteine
Km (mM)
kcat (s1)
1.60 1.78 0.20
2.90 1.67 0.03
kcat/Km (s1mM1) 1.81 0.94 0.15
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
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FIG. 2. Phylogenic tree of g-family enzymes. Figure was drawn using ClustalW. OAHS_Tt, O-acetyl-L-homoserine sulfhydrylase from Thermus thermophilus; OAHS_Pp, O-acetyl-Lhomoserine sulfhydrylase from Pseudomonas putida; OSHS_Pp, O-succinyl-L-homoserine sulfhydrylase from P. putida; CBL_Sc, cystathionine b-lyase from Saccharomyces cerevisiae; CBL_Ll, cystathionine b-lyase from Lactococcus lactis; CBL_At, cystathionine b-lyase from Arabidopsis thaliana; CBL_Ec, cystathionine b-lyase from Escherichia coli; CGS_Nt, cystathionine g-synthase from Nicotiana tabacum; CGS_At, cystathionine g-synthase from A. thaliana; CGS_Ec, cystathionine g-synthase from E. coli; MGL_Bl, L-methionine g-lyase from Brevibacterium linens; MGL_Cf, L-methionine g-lyase from Citrobacter freundii; MGL_Eh, L-methionine g-lyase from Entamoeba histolytica; MGL_Pp, L-methionine g-lyase from P. putida; MGL_Td, L-methionine g-lyase from Treponema denticola; MGL_Tv, L-methionine g-lyase from Trichomonas vaginalis; MGL_At, L-methionine g-lyase from A. thaliana; MGL_Sa, L-methionine g-lyase from S. avermitilis.
MGL_Sa from S. avermitilis formed a cluster with the sulfhydrylase group and was closer to the cystathionine g-synthase and cystathionine b-lyase than MGL from P. putida. Then, substrate specificity for some elimination substrates was investigated (Table S1). The g-elimination activities toward g-elimination substrates, DL-homocysteine and O-succinyl-L-homoserine, were lower than that of L-methionine. The data for the b-elimination activities toward cystathionine, L-cysteine, S-methyl-L-cysteine, Sethyl-L-cysteine, O-phospho-L-serine, O-acetyl-L-serine and Oacetyl-L-serine showed a similar tendency like as MGL_Pp (15). Furthermore, it was found that MGL_Sa has lower g-replacement activities than those of MGL_Pp by TLC analysis (Fig. S2). Cystathionine synthesis reaction of MGL_Sa and MGL_Pp was carried out under the condition as follows: 20 mL substrate solution in 100 mM KPB (pH 8.0) with 100 mM O-succinyl-L-homoserine, 100 mM L-cysteine and 10 mM PLP was prepared, 5 mL enzyme solutions were added, respectively, and these were reactioned at 37 C for 1 h. These were stopped by adding 2.5 mL of 50% TCA. Cystathionine synthesis reaction of cystathionine g-synthase from Sulfolobus tokodaii (CGS_St) was carried out under the condition as follows: 20 mL substrate solution in 100 mM TriseHCl (pH 9.0) with 100 mM O-succinyl-L-homoserine, 100 mM L-cysteine and 10 mM PLP was prepared, 5 mL enzyme solution was added and it was reactioned at 70 C for 1 h. It was stopped by adding 2.5 mL of 50% TCA. S-(b-hydroxyethyl) homocysteine synthesis reaction of MGL_Sa and MGL_Pp was carried out under the same conditions in cystathionine synthesis, replacing L-cysteine into 160 mM 2mercaptoethanol (18). S-(b-hydroxyethyl) homocysteine synthesis reaction of CGS_St was also carried out under the same
conditions in cystathionine synthesis, replacing L-cysteine into 160 mM 2-mercaptoethanol. These data showed that SAV7062 has no replacement activity for cystathionine and S-(b-hydroxyethyl) homocysteine synthesis, indicating SAV7062 is L-methionine glyase and interestingly has higher reaction specificity than MGL_Pp. For the phylogenetic analysis it was found that this protein was clearly separated from heterologous MGLs in the early stages of evolution and was nearly similar to plant MGLs (Fig. 2). This report was mainly about MGLs from Streptomycete and was not only concerned with phylogenic trees but also MGL evolution. In this study the enzyme activity of SAV7062 as a MGL involved in S. avermitilis sulfur metabolism was clarified partially, however there are little data to speculate the physiological roles of this enzyme in Streptomycete. We wish to keep in analyzing the sulfur metabolism in Streptomycete to clarify the mechanism the secondary metabolism. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.02.019 References 1. Nakayama, T., Esaki, N., Sugie, K., Beresov, T. T., Tanaka, H., and Soda, K.: Purification of bacterial L-methionine g-lyase, Anal. Biochem., 138, 421e424 (1984). 2. Nakayama, T., Esaki, N., Lee, W. J., Tanaka, I., Tanaka, H., and Soda, K.: Purification and properties of L-methionine g-lyase from Aeromonas sp. Agric. Biol. Chem., 48, 2367e2369 (1984). 3. Manukhov, I. V., Mamaeva, D. V., Rastorguev, S. M., Faleev, N. G., Morozova, E. A., Demidkina, T. V., and Zavilgelsky, G. B.: A gene encoding Lmethionine g-lyase is present in Enterobacteriaceae family genomes:
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019
4
4.
5.
6.
7. 8.
9.
10.
11.
KUDOU ET AL. identification and characterization of Citrobacter freundii L-methionine g-lyase, J. Bacteriol., 187, 3889e3893 (2005). Amarita, F., Yvon, M., Nardi, M., Chambellon, E., Delettre, J., and Bonnarme, P.: Identification and functional analysis of the gene encoding methionine g-lyase in Brevibacterium linens, Appl. Environ. Microbiol., 70, 7348e7354 (2004). Yoshimura, M., Nakano, Y., Yamashita, Y., Oho, T., Saito, T., and Koga, T.: Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis, Infect. Immun., 68, 6912e6916 (2000). Fukamachi, H., Nakano, Y., Okano, S., Shibata, Y., Abiko, Y., and Yamashita, Y.: High production of methyl mercaptan by L-methionine-a-deamino-g-mercaptomethane lyase from Treponema denticola, Biochem. Biophys. Res. Commun., 331, 127e131 (2005). Lockwood, B. C. and Coombs, G. H.: Purification and characterization of methionine g-lyase from Trichomonas vaginalis, Biochem. J., 279, 675e682 (1991). Tokoro, M., Asai, T., Kobayashi, S., Takeuchi, T., and Nozaki, T.: Identification and characterization of two isoenzymes of methionine g-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways, J. Biol. Chem., 278, 42717e42727 (2003). Rébeillé, F., Jabrin, S., Bligny, R., Loizeau, K., Gambonnet, B., Van Wilder, V., Douce, R., and Ravanel, S.: Methionine catabolism in Arabidopsis cells is initiated by a g-cleavage process and leads to S-methylcysteine and isoleucine syntheses, Proc. Natl. Acad. Sci. USA, 103, 15687e15692 (2006). Marvin, D. S., Delia, V., and Otto, H.: Biosynthesis of the avermectins by Streptomyces avermitilis, incorporation of labeled precursors, J. Antibiot. (Tokyo), 39, 541e549 (1986). Yoon, Y., Younghee, P., Yong, S. Y., Youngshim, L., Geunhyeong, J., Jun, C. P., Joong, H. A., and Yoongho, L.: Characterization of an O-methyltransferase
J. BIOSCI. BIOENG.,
12.
13.
14.
15.
16.
17.
18.
from Streptomyces avermitilis MA-4680, J. Microbiol. Biotechnol., 20, 1359e1366 (2010). Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M., and Omura, S.: Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis, Nat. Biotechnol., 21, 526e531 (2003). Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takakura, T., Yoshioka, T., Yagi, S., Hoffman, R. M., Takimoto, A., Esaki, N., and Inagaki, K.: Structure of the antitumour enzyme L-methionine g-lyase from Pseudomonas putida at 1.8 Å resolution, J. Biochem., 141, 535e544 (2007). Inoue, H., Inagaki, K., Adachi, N., Tamura, T., Esaki, N., Soda, K., and Tanaka, H.: Role of tyrosine 114 of L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 64, 2336e2343 (2000). Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Esaki, N., and Inagaki, K.: The role of cysteine 116 in the active site of the antitumor enzyme L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 72, 1722e1730 (2008). Takakura, T., Mitsushima, K., Yagi, S., Inagaki, K., Tanaka, H., Esaki, N., Soda, K., and Takimoto, A.: Assay method for antitumor L-methionine g-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine, Anal. Biochem., 327, 233e240 (2004). Johnston, M., Jankowski, D., Marcotte, P., Tanaka, H., Esaki, N., Soda, K., and Walsh, C.: Suicide inactivation of bacterial cystathionine g-synthase and methionine g-lyase during processing of L-propargylglycine, Biochemistry, 18, 4690e4701 (1979). Harada, S., and Inagaki, K.: The role of amino acids residues in the active site of L-methionine g-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 76, 1275e1284 (2012).
Please cite this article in press as: Kudou, D., et al., Molecular cloning and characterization of L-methionine g-lyase from Streptomyces avermitilis, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.019