Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Makoto Nishiyama
umanis{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AB097117.
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INTRODUCTION |
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In lower eukaryotes, the enzyme -aminoadipate aminotransferase (AAAAT; EC 2.6.1.39), which catalyses the transamination reaction using 2-oxoadipate (2-OA) as an amino acceptor to yield AAA, is suggested to be involved in lysine biosynthesis. Matsuda & Ogur (1969)
isolated and characterized glutamate : 2-OA aminotransferases 1 and 2 from Saccharomyces cerevisiae and reported, based on the catalytic properties, that enzymes 2 and 1 were biosynthetic and catabolic enzymes, respectively, in lysine metabolism. However, to date, the corresponding genes have not been identified in S. cerevisiae or other organisms. To elucidate the whole pathway of lysine biosynthesis in T. thermophilus and the evolutionary relationships between lysine biosynthesis and other metabolic pathways, including leucine biosynthesis, arginine biosynthesis and the TCA cycle, we attempted to clone the gene from the micro-organism and analyse its function.
In this paper, we describe the cloning of a gene (lysN) encoding a mammalian kynurenine/AAAAT homologue, which is presumably involved in lysine biosynthesis in T. thermophilus HB27. We also report the kinetic properties of LysN, which shows AAAAT activity and branched-chain amino acid aminotransferase activity.
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METHODS |
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All the chemicals were purchased from Sigma or from Kanto Chemicals. NAD+-dependent glutamate dehydrogenase was purchased from Toyobo. Enzymes for DNA manipulation were purchased from Takara Shuzo. Oligonucleotide primers used in this study are listed in Table 1. DNA manipulation was performed according to the methods of Sambrook & Russell (2001)
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Disruption of lysN in T. thermophilus HB27.
To introduce an XbaI site in the lysN gene, PCRs were performed using two pairs of oligonucleotides as primers, A3AT-1/A3AT-2 and A3AT-3/3AT-4. After appropriate treatment with restriction endonucleases, the two amplified fragments were inserted into pUC18 to generate pUC18/A3AT. The 1·1 kb DNA fragment encoding the thermostable kanamycin resistance gene (Hoseki et al., 1999) was amplified as the XbaI fragment by PCR, using the oligonucleotides HTK-1 and HTK-2 as primers, and inserted into pUC18/A3AT at the XbaI site to yield pUCATKmR. Disruption of the chromosomal copy of lysN was performed by using pUCATKmR as described previously (Hidaka et al., 1994
; Koyoma et al., 1986
), and confirmed by PCRs using the oligonucleotides A3AT-1/AAA3 and A3AT-1/A3AT-5 as primers.
Phenotypic analysis of lysN mutant.
The lysN knockout mutant of T. thermophilus was cultured in 1 ml TM medium overnight. TM medium contains 0·8 % polypeptone, 0·4 % Bacto Yeast Extract, 0·2 % NaCl, 0·4 mM CaCl2, and 0·4 mM MgCl2. After centrifugation of the culture at 20 000 g for 1 min, the precipitate was washed with minimal medium (MP medium; Kobashi et al., 1999; Tanaka et al., 1981
) four times and resuspended in 1 ml MP medium. A 10 µl volume of the resuspended culture was added to 5 ml MP medium and incubated at 70 °C. Growth of the cells was analysed by monitoring the OD600 of the cell culture at appropriate intervals.
Purification of recombinant LysN from E. coli.
E. coli BL21-Codon-Plus (DE3)-RIL cells harbouring pETLysN7 were cultured in 800 ml of 2x YT medium containing 50 µg kanamycin ml1 and 30 µg chloramphenicol ml1. When the culture had reached an OD600 of 0·5, IPTG (final concentration, 0·1 mM) was added. The culture was continued at 25 °C for an additional 12 h after the induction. E. coli(pETLysN7) cells (wet weight, 4·5 g) collected from the 800 ml culture were suspended in 27 ml buffer I (20 mM potassium phosphate buffer, pH 6·5, 0·5 mM EDTA) and disrupted by sonication. The supernatant prepared by centrifugation at 40 000 g for 20 min was heated at 80 °C for 20 min, and denatured proteins from E. coli cells were removed by centrifugation at 40 000 g for 20 min. Supernatant fractions were applied to an anion-exchange column (DE-52; Whatman), pre-equilibrated with buffer I and eluted with buffer I containing 0·1 M NaCl. After the addition of ammonium sulfate to a final concentration of 65 % saturation to active fractions, the resultant precipitate was collected by centrifugation at 40 000 g for 30 min. The precipitated proteins were solubilized with buffer I and applied onto a Hi-load 26/60 Superdex 200 prep-grade column (Amersham-Pharmacia Biotech) equilibrated with buffer I containing 0·2 M NaCl to yield the purified preparation. Protein concentration was determined by the method of Bradford (1976) using a protein assay kit (Nippon Bio-Rad).
Sedimentation equilibrium analysis.
Sedimentation equilibrium analysis of purified LysN was performed with a Beckman Optima XL-A analytical ultracentrifuge fitted with a Beckman An-60Ti analytical rotor. The subunit organization of the enzyme was analysed according to the procedure of Van Holde & Baldwin (1958).
Enzyme assays.
Enzyme assays were carried out according to the method described by Nakatani et al. (1970). The temperature for the assay was set to 45 °C to avoid non-enzymic oxidation of NADH. The enzyme reaction was initiated by adding 10 µl of the enzyme solution (0·25 mg ml1) to 1 ml reaction buffer (100 mM potassium phosphate buffer, pH 7·5, 50 mM NH4Cl, 10120 µM 2-OA, 2·0 mM L-glutamate, 0·15 mM NADH, 10 µM pyridoxal 5'-phosphate, 11·9 U glutamate dehydrogenase ml1), which was preincubated at 45 °C for 5 min. For measuring the activity of 2-oxoisocaproate (2-OIC), 2-oxoisovalerate (2-OIV) and 2-oxo-3-methylvalerate (2-OMV), 10200 µM 2-OIC, 10120 µM 2-OIV or 0·15 mM 2-OMV was added to the reaction buffer instead of 2-OA. The reaction was monitored at 45 °C by measuring the decrease in absorption at 340 nm. Kinetic parameters were calculated by using an initial velocity program, HYPER (Cleland, 1979
). Specific activity was determined by using 20 mM L-glutamate as the amino donor, and 1 mM 2-OA, 2-OIC, 2-OIV or phenylpyruvate, or 10 mM 2-OMV, pyruvate or oxaloacetate as the amino acceptor. One unit of enzyme activity was defined as the amount of enzyme that reduced 1 µmol NADH min1 at 45 °C in the reaction.
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RESULTS |
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By searching in the literature for the enzyme that catalyses the transamination reaction, we found that the aminotransferase Kat2 (EC 2.6.1.7), which irreversibly transaminates a tryptophan metabolite, kynurenine, to form kynurenic acid, a putative endogenous modulator of the glutamatergic neurotransmitter in mammals, catalyses the transamination reaction between 2-OA and glutamate to produce AAA and 2-oxoglutarate (Rico et al., 1995), although mammals cannot synthesize lysine. We then analysed the distribution of Kat2 homologues in micro-organisms and found that all the micro-organisms that have been suggested to possess the lysine biosynthesis pathway through AAA carry a gene encoding a protein that shows considerable identity to Kat2 in amino acid sequence: YGL202W (32 %) from S. cerevisiae, DR1588 (31 %) from Deinococcus radiodurans, PH0207 (31 %) from Pyrococcus horikoshii, ST1411 (32 %) from Sulfolobus tokodaii and APE0169 (30 %) from Aeropyrum pernix. We therefore decided to clone a Kat2 homologue from T. thermophilus. Determination of the nucleotide sequence of the cloned full-length Kat2 homologue revealed that the gene encodes a protein composed of 397 amino acid residues with a predicted molecular mass of 43 845 Da. The Kat2 homologue showed significant identity in amino acid sequence to DR1588 (44 %), PH0207 (35 %), ST1411 (37 %) and APE0169 (30 %).
Disruption of Kat2 homologue in T. thermophilus
We next investigated the role of the Kat2 homologue in T. thermophilus. For this purpose, we constructed a mutant of T. thermophilus with disruption in the chromosomal Kat2 homologue as described in Methods. The Kat2-homologue disruptant, TM1001, required longer lag time for growth in the shift-down experiments from rich medium to minimal MP medium: the lag time was about 20 h for the mutant and 10 h for the wild-type (Fig. 1). The extended lag phase was significantly reversed by addition of 0·1 mM lysine or AAA, but not affected by addition of 0·1 mM 2-OA. Furthermore, the growth rate of the mutant (doubling time, 190 min) was found to be lower than that of the wild-type strain (84 min) in minimal medium. The doubling time of the mutant was not shortened by addition of 2-OA (192 min), but it was significantly improved by addition of AAA (121 min) and lysine (151 min). These results suggested the involvement of the Kat2 homologue in lysine biosynthesis of T. thermophilus. Hereafter, we refer to this gene as lysN.
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Catalytic properties of LysN
For an initial evaluation of the substrate specificity of LysN, we determined specific activities for several compounds in the crude extract prepared by heat treatment of the E. coli cell lysate and successive centrifugation steps. As expected, T. thermophilus LysN showed transamination of 2-OA to produce AAA with a specific activity of 6788 U (mg protein)1 (Table 2). When similar analysis was done in the reverse reaction at the same pH with AAA and 2-oxoglutarate as the amino donor and the amino acceptor, respectively, only negligible activity was detected. From this analysis, we concluded that LysN was a biosynthetic enzyme for lysine biosynthesis. We next analysed the transamination reaction with several 2-oxoacids as the amino acceptor by using glutamate as an amino donor; we found that LysN recognized all these compounds as a substrate.
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DISCUSSION |
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The addition of AAA or lysine to the minimal MP medium indeed shortened lag phase for growth of the lysN-knockout mutant and improved its growth rate, but it did not completely reverse the extended lag and the slow growth. This observation may suggest some other roles of LysN that are required for the normal growth of the cells as suggested in the case of Aro8p (Urrestarazu et al., 1998; Zabriskie & Jackson, 2000
). We would like to note that the expression of the lysN gene is not regulated by lysine in T. thermophilus cells (data not shown), which is in contrast to many other genes involved in amino acid biosynthesis. This might be evidence, though indirect, suggesting the possible involvement of LysN in several unrelated metabolic pathways. It is noteworthy that LysN recognizes 2-OIC more efficiently than oxaloacetate, as is the case for BcAT, which is involved in biosynthesis of branched-chain amino acids, although no significant identity in amino acid sequence has been found between LysN and BcAT (data not shown). Thus, since LysN has aminotransferase activity not only for AAA, but also for the branched-chain amino acids, there is the possibility that LysN might be involved at least in both lysine and branched-chain amino acid biosyntheses. There is a unique aminotransferase group, COG1167, in which a GntR-type DNA-binding HTH helix domain is fused at the N-terminus of type I aminotransferase. T. thermophilus AAAAT also belongs to the COG1167 group, but it lacks the N-terminal GntR-type HTH domain as found in normal aminotransferases. Therefore, T. thermophilus LysN does not function as a transcription regulator. However, it should be noted that the corresponding protein of Corynebacterium glutamicum, PdxR (cg0897), is involved in valine biosynthesis as well as pyridoxine biosynthesis (McHardy et al., 2003
), which suggests a functional relationship between LysN and the MocR-type aminotransferase in that both the proteins play a putative role in branched-chain amino acid biosynthesis. In any case, such broad substrate specificity is a typical feature found in the enzymes involved in other reactions in lysine biosynthesis (Miyazaki et al., 2001
, 2002
, 2003
; Wulandari et al., 2002
). It should be noted that addition of branched-chain amino acids, isoleucine, leucine and valine, remarkably accelerated the growth of T. thermophilus cells in minimal medium and no difference was detected in growth between the wild-type and the lysN mutant (data not shown), suggesting that the amino group of the branched-chain amino acid might be, directly or indirectly, transferred to 2-OA to form AAA. Considering the similarity in amino acid sequence between LysN and YGL202W (ARO8), it might be possible to consider that LysN is involved in aromatic amino acid biosynthesis; however, this is unlikely because the activity of LysN for phenylpyruvate was quite low (Table 2
) and addition of both phenylalanine and tyrosine had no effect on the growth of the T. thermophilus mutant lacking the lysN gene in the minimal medium (data not shown). To further elucidate the role of LysN in Thermus cells, detailed analysis of the nutrition required for the normal growth of the mutant will be necessary.
On the other hand, the Pyrococcus furiosus aminotransferase, PF0121, a possible orthologue of LysN and PH0207 of P. horikoshii, has been characterized biochemically and referred to as aromatic amino acid aminotransferase (Andreotti et al., 1995; Schut et al., 2003
). Expression of PF0121 was upregulated in peptide-grown cells, suggesting its role in catabolism in the archaeon (Schut et al., 2003
). On the other hand, we observed that the catabolic activity of LysN to convert AAA to 2-OA was negligible and addition of lysine had no effect on the expression profile of lysN in T. thermophilus (data not shown). These results could suggest that LysN plays a role that is different from that of PF0121 in P. furiosus. Nevertheless, it is of interest to analyse the substrate specificity of PF0121 for these amino acids.
In this study, we have shown that LysN catalyses the fourth reaction of lysine biosynthesis through the AAA pathway. Detailed analysis of the mechanism used by LysN to recognize several compounds as substrates may provide a clue to elucidate the evolution of aminotransferases. For this purpose, it is necessary to determine the three-dimensional structure of LysN and the complexes it forms with substrate analogues.
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ACKNOWLEDGEMENTS |
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Received 15 January 2004;
revised 18 March 2004;
accepted 2 April 2004.
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