N-Methyl-L-amino acid dehydrogenase from Pseudomonas putida A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily Hisaaki Mihara 1, *, Hisashi Muramatsu 1, *, Ryo Kakutani 1 , Mari Yasuda 2 , Makoto Ueda 2 , Tatsuo Kurihara 1 and Nobuyoshi Esaki 1 1 Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan 2 Yokohama Research Center, Mitsubishi Chemical Corp., Yokohama, Japan n-Methyl amino acids occur naturally as components of several depsipeptides, including cyclosporin A [1], vancomycin, destruxins [2], didemnins [3], dolastatin [4], and enniatin [5], as well as a calpain inhibitor from Streptomyces griseus [6] and a pilin from Pseudomonas aeruginosa [7]. Enniatins consist of alternating residues of d-2-hydroxyisovalerate and N-methyl-l-valine (or N-methyl-l-isoleucine) [5]. N-Methylphenylalanine is the N-terminal amino acid of pilins of most bacterial pathogens expressing type IV pili [7]. The synthesis of these N-methylated peptides is catalyzed by specific N-methyltransferases such as enniatin synthetase [8] and PilD [9] using S-adenosyl-l-methionine as the methyl group donor. Free forms of N-methyl amino acids also occur in bivalves [10,11], several plant species [12], and some bacteria [13,14]. In Aminobacter aminovorans (formerly Pseudomonas MA) [15], N-methylglutamate and Keywords methylamine; NADPH; N-methyl- L-amino acid dehydrogenase; N-methyl- L-amino acid; Pseudomonas putida Correspondence N Esaki, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Fax: 81 774 38 3248 Tel: 81 774 38 3240 E-mail: esaki@scl.kyoto-u.ac.jp *Note Equivalent first authors. Database Nucleotide sequence data reported are avail- able in the DDBJ ⁄ EMBL ⁄ GenBank databas- es under the accession number AB190215. (Received 8 October 2004, revised 12 December 2004, accepted 22 December 2004) doi:10.1111/j.1742-4658.2004.04541.x We found N-methyl-l-amino acid dehydrogenase activity in various bacter- ial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-l-amino acids from the corresponding a-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a sub- strate, and the enzyme was clearly distinct from conventional NAD(P)- dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases. Abbreviation NMAADH, N-methyl- L-amino acid dehydrogenase. FEBS Journal 272 (2005) 1117–1123 ª 2005 FEBS 1117 ammonia are formed from methylamine and glutamate by N-methylglutamate synthase (EC 2.1.1.21) [16]. Another A. aminovorans strain, formerly called Pseudo- monas MS, produces N-methylalanine from methyl- amine and pyruvate in the presence of NADPH with N-methylalanine dehydrogenase (EC 1.4.1.17) [17]. Therefore, in contrast with the N-methyl groups of depsipeptides and pilins, those of free N-methyl amino acids are derived from methylamine. The reaction catalyzed by N-methylalanine dehydro- genase resembles that of alanine dehydrogenase (EC 1.4.1.1) [18], which acts on ammonia instead of meth- ylamine. However, their mechanisms for discriminating between methylamine and ammonia are poorly under- stood. N-Methyl amino acids are structurally similar to opines such as nopaline, octopine, lysopine, and histopine, which are N-substituted amino acids found in crown gall tumor tissues induced by infection with Agrobacterium tumefaciens [19,20]. d-Octopine dehy- drogenase (EC 1.5.1.11) [21], alanopine dehydrogenase (EC 1.5.1.17) [22] and d-nopaline dehydrogenase (EC 1.5.1.19) [23], involved in the metabolism of opines, have been characterized and shown to be similar to NAD(P)-dependent amino acid dehydrogenases [24]. Dairi & Asano [25] have shown that the primary struc- ture of the opine dehydrogenase from Arthrobacter sp. strain 1C is similar to d-lysopine dehydrogenase (EC 1.5.1.16), d-nopaline dehydrogenase, and phenylalan- ine dehydrogenase (EC 1.4.1.20). They share the gly- cine-rich nucleotide-binding motif, which is strongly conserved among NAD(P)-dependent dehydrogenases [24,26]. We have screened bacterial strains that produce an enzyme catalyzing the production of N-methyl-l- phenylalanine from phenylpyruvate and methylamine in order to develop a new method for the industrial production of N-methyl-l-phenylalanine. We found that Pseudomonas putida ATCC12633 shows high N-methyl-l-phenylalanine dehydrogenase activity [27]. We here describe the gene cloning and characterization of the enzyme, which belongs to a protein superfamily completely different from that of conventional amino acid dehydrogenases and opine dehydrogenases. Results and Discussion Identification of a gene encoding N-methyl-L- amino acid dehydrogenase (NMAADH) We tested about 100 bacterial strains for the ability to form N-methylphenylalanine from phenylpyruvate and methylamine. We found such activity in several strains, including P. putida, Bacillus alvei, Brevibacterium sac- chrolyticum, Brevibacterium linens, Agrobacterium viscosum, Aerobacter aerogenes, P. aeruginosa, and P. fluorescens. The crude extract from P. putida ATCC12633 exhibited the highest activity, and this strain was chosen for further studies. We cultivated the P. putida strain in 200 L Luria– Bertani medium and obtained about 2.5 kg wet cells. NMAADH was purified 150-fold with a yield of 0.066% by purification steps with such chromatogra- phy columns as SuperQ-Toyopearl, Butyl-Toyopearl, DEAE-Toyopearl, Green-Sepharose, RESOURCE PHE, and Blue-Sepharose. SDS ⁄ PAGE analysis and a TLC-based assay of fractions from the Blue-Sepharose column revealed that a 36-kDa protein was the enzyme exhibiting the NMAADH activity. The N-terminal amino-acid sequence of this protein was determined to be XAPSTSTVVRVPFTEL. We carried out a blast search using the unfinished microbial genome database at TIGR (http://www.tigr.org/tdb/) with this sequence and found that it was very similar to that of a putative protein PP3591 (NCBI database number, AAN69191) encoded by the genome of P. putida KT2440. PP3591 contained 341 amino-acid residues with a calculated molecular mass of 35 972.9 Da and showed moderate sequence similarity to malate dehydrogenase homo- logs. Cloning and sequencing of the gene encoding NMAADH A DNA fragment containing a gene for NMAADH, named dpkA, from P. putida ATCC12633 was obtained by PCR with primers designed on the basis of sequences of PP3591 from P. putida KT2440. The nucleotide sequence of the cloned gene (1026 bp; 341 amino acids; molecular mass, 35 972.8 Da) matched completely that directly sequenced with the chromo- somal DNA of P. putida ATCC12633: no nucleotide substitution occurred on PCR (DDBJ ⁄ EMBL ⁄ GenBank accession number: AB190215). The nucleo- tide and deduced amino-acid sequences of the gene showed high identities (97.9% and 99.1%, respectively) with those of the PP3591 gene of P. putida KT2440. Purification and subunit structure of NMAADH The NMAADH gene was inserted into pET21a(+) to yield the plasmid pDPKA. NMAADH was purified to homogeneity by chromatography using a Green-Seph- arose and a DEAE-Toyopearl column from the recom- binant Escherichia coli BL21(DE3) cells carrying pDPKA with a yield of 23%. The N-terminal amino- acid sequence of the purified enzyme was identical with N-Methyl-L-amino acid dehydrogenase from P. putida H. Mihara et al. 1118 FEBS Journal 272 (2005) 1117–1123 ª 2005 FEBS the deduced amino-acid sequence of the enzyme, except that the initial Met was removed. The initial Met was also missing in the enzyme purified from P. putida ATCC12633. The purified enzyme gave a single band with a molecular mass of 37 kDa on SDS ⁄ PAGE (Fig. 1). The molecular mass of the native enzyme was found to be 74 kDa by gel filtration. Therefore, the enzyme probably consists of two identical subunits. Substrate specificity and effects of various compounds on the enzymatic activity The substrate specificity of the enzyme in NADPH- dependent reductive amination was examined with various a-oxo acids and amines. Pyruvate was the best substrate of the various a-oxo acids tested (Table 1). The enzyme also acted on a-oxohexanoate, phenylpyru- vate, a-oxobutyrate, fluoropyruvate, a-oxovalerate, a-oxoisocaproate, a-oxo-octanoate, and hydroxypyru- vate. However, branched-chain a-oxo acids, such as a-oxoisovalerate and a-oxo-b-methylvalerate, were inert. The best substrate was methylamine, but the enzyme also showed activity towards ethylamine (4.4%, relative to methylamine), 2-chloroethylamine (0.74%), 2-bromoethylamine (0.27%), n-propylamine (0.16%) and dimethylamine (0.12%). Weak activities (0.06– 0.03%, relative to methylamine) were found with ethylenediamine, hydroxylamine, isopropylamine, n- butylamine, n-amylamine, n-hexylamine, 1,6-diamino- hexane, and spermidine. Interestingly, the enzyme was unable to use ammonia as a substrate and was distinct from alanine dehydrogenase [18] and N-methylalanine dehydrogenase found by Lin & Wagner [17]. Lysine and ornithine were inert as an amine substrate; the enzyme shows no opine dehydrogenase activity with these amino acids. The enzyme was sensitive to bivalent metal ions, such as CuCl 2 , HgCl 2 , and CoCl 2 . Concen- trations of the enzyme causing 50% inhibition were 0.35 lm for CuCl 2 , 0.96 lm for HgCl 2 , and 160 lm for CoCl 2 . Carbonyl reagents, including hydroxylamine, phenylhydrazine, semicarbazide, and aminoguanidine, did not inhibit the enzyme. Chelating agents, such as o-phenanthroline and EDTA, hardly inhibited the enzyme. Thiol reagents, such as N-ethylmaleimide and iodoacetate, and a serine-modifying reagent, phenyl- methanesulfonyl fluoride, did not affect the enzyme. Non-substrate a-oxo acids, a-oxoisovalerate and a-oxo- b-methylvalerate, did not inhibit the enzyme. Other enzymological properties The enzyme showed maximum activity at pH 10.0 for both reductive amination of phenylpyruvate and oxi- dative deamination of N-methyl-l-alanine. It was sta- ble between pH 6.0 and 10.0 and showed maximum activity at 35 °C. However, it was unstable at this tem- perature and lost 30% of its original activity after a 30-min incubation in 20 mm Tris ⁄ HCl buffer at pH 7.0. The enzyme was stable at temperatures below 30 °C for at least 30 min and used both NAD + and NADH as coenzymes. However, the specific activity of the enzyme with NADPH (42 UÆmg )1 ) was more than 300 times higher than with NADH (0.13 UÆmg )1 )in reductive amination of pyruvate. Oxidative deamina- tion of N-methyl-l-alanine with NADP + (0.36 UÆ mg )1 ) was markedly lower than reductive amination of pyruvate with NADPH. Fig. 1. Overexpression and purification of recombinant NMAADH. Protein samples from various stages of the purification were sub- jected to SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. Lane 1, marker proteins (sizes in kDa are shown); lane 2, crude extract; lane 3, after the Green-Sepharose CL-4B chromatography; lane 4, after the DEAE-Toyopearl chromatography. Table 1. Substrate specificity of NMAADH in the forward reaction. NMAADH activity was determined in reaction mixture containing 10 m M a-oxo acid, 60 mM methylamine (pH 10.0), and 0.2 m M NADPH at 30 °C. Substrate Relative activity (%) Pyruvate 100 a-Oxohexanoate 52 Phenylpyruvate 30 a-Oxobutyrate 30 Fluoropyruvate 27 a-Oxovalerate 16 a-Oxoisocaproate 9.1 a-Oxo-octanoate 7.6 Hydroxypyruvate 5.8 H. Mihara et al. N-Methyl- L-amino acid dehydrogenase from P. putida FEBS Journal 272 (2005) 1117–1123 ª 2005 FEBS 1119 Kinetic studies The kinetic mechanism of NMAADH was studied by steady-state kinetic analyses. NADPH-dependent methylamination of pyruvate showed hyperbolic sat- uration curves and gave linear double-reciprocal plots in the range 1–10 mm pyruvate, 5–60 mm methyl- amine, and 0.05–0.3 mm NADPH. The families of lines obtained from the double-reciprocal plots of reac- tion velocities against pyruvate concentrations at var- ious fixed concentrations of methylamine intersected to the left of the 1 ⁄ v axis. Similar results were obtained with various concentrations of NADPH and fixed con- centrations of pyruvate. These indicate a sequential mechanism for the NMAADH reactions. In fact, the data obtained gave a good global fit to the equation of an ordered Ter Bi mechanism. To test the kinetic mechanism further, we performed inhibition studies with both products of the reaction, NADP + and N-methyl-l-alanine. At a fixed subsaturat- ing concentration of N-methyl-l-alanine (100 mm), the inhibition by NADPH was competitive linearly with respect to NADP + . With N-methyl-l-alanine as the variable substrate, at a fixed subsaturating concentra- tion of NADP + (0.2 mm), NADPH was a linear mixed- type inhibitor. Pyruvate behaved as a noncompetitive inhibitor with regard to NADP + and N-methyl-l-alan- ine. Methylamine behaved as a mixed-type inhibitor with respect to NADP + and N -methyl-l-alanine. These results indicate that NADPH-dependent N -methyl-l- alanine formation proceeds in an ordered sequential Ter Bi mechanism, in which NADPH, pyruvate, and meth- ylamine are bound to the enzyme in this order, and N-methyl-l-alanine and then NADP + are released from the enzyme. Therefore, the kinetic mechanism of the enzyme is the same as those of conventional NAD(P)- dependent amino acid dehydrogenases [25–27]. Comparison with other NAD(P)-dependent dehydrogenases The reaction catalyzed by NMAADH is similar to those by conventional NAD(P)-dependent amino acid dehydrogenases, such as glutamate dehydrogenase [28], leucine dehydrogenase [29], and phenylalanine dehy- drogenase [30], as well as opine dehydrogenase [25,31], which have a common structure called the Rossmann fold for their NAD(P)-binding sites [26]. However, NMAADH shows little (< 15%) sequence similarity to the NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases and has no sequence motif char- acteristic of the Rossmann fold, indicating that the structure of the nicotinamide nucleotide-binding site of NMAADH is completely different from those with the Rossmann fold. A homology search revealed that NMAADH belongs to a new protein superfamily including NAD(P)-dependent malate ⁄ l-lactate dehy- drogenases with no sequence homology to conventional malate dehydrogenases or lactate dehydrogenases [32]. The superfamily contains, in addition to the malate ⁄ l-lactate dehydrogenases, various NAD(P)-dependent dehydrogenases, such as l-sulfolactate dehydrogenase [33], ureidoglycolate dehydrogenase [34], and 2,3- diketo-l-gulonate reductase [35]. The action on a-oxo acids as substrate is common to all these enzymes, and NMAADH is a new addition to this superfamily. Conclusion We have identified an enzyme catalyzing the NADPH- dependent formation of N -methyl-l-phenylalanine from phenylpyruvate and methylamine. The enzyme is unique in that it does not act on ammonia at all and shows broad specificity for various a-oxo acids. Accord- ingly, we named the enzyme N-methyl-l-amino acid dehydrogenase to distinguish it from the previously reported N-methylalanine dehydrogenase [17]. More- over, this study shows that the enzyme belongs to a new NAD(P)-dependent oxidoreductase family and is struc- turally distinct from conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases. Experimental procedures Materials N-Methyl-l-phenylalanine and d-amino acid oxidase from porcine kidney were purchased from Sigma (St Louis, MO, USA). RESOURCE PHE, Superose 12, Sepharose CL-4B, and molecular-mass marker proteins were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). SuperQ- Toyopearl, DEAE-Toyopearl, and Butyl-Toyopearl were from Tosoh (Tokyo, Japan). Green-Sepharose was prepared as described previously [36]. NADH, NADPH, and mole- cular-mass marker proteins for gel filtration were from Oriental Yeast (Tokyo, Japan). Restriction enzymes and kits for genetic manipulation were from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), and New England Biolabs (Beverly, MA, USA). All other reagents were of analytical grade from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan). Culture and screening of bacteria Bacterial strains were cultivated for 15–20 h at 30 °Cin 0.5 mL screening medium containing citric acid (0.5 gÆL )1 ), N-Methyl-L-amino acid dehydrogenase from P. putida H. Mihara et al. 1120 FEBS Journal 272 (2005) 1117–1123 ª 2005 FEBS N-methyl-l-phenylalanine (1 gÆL )1 ), glucose (2 gÆL )1 ), K 2 HPO 4 (7 gÆL )1 ), KH 2 PO 4 (3 gÆL )1 ), and MgSO 4 Æ7H 2 O (0.1 gÆL )1 ) (pH 6.9). The cells were harvested by centrifu- gation, resuspended in 0.1 mL reaction mixture containing 5mm calcium phenylpyruvate and 1 m methylam- ine ⁄ H 2 SO 4 (pH 8.9), and incubated for 3 h at 30 °C. The formation of N-methylphenylalanine in the reaction mix- ture was analyzed by TLC on a silica gel 60 plate (Merck, Darmstadt, Germany) or by HPLC with an Ultron ES-PhCD column (Shinwa Kako, Kyoto, Japan). The sol- vent system for TLC was ethyl acetate ⁄ ethanol ⁄ acetic acid ⁄ water (5 : 2 : 1 : 1, by vol.), and N-methylphenyl- alanine was visualized with a coloring reagent containing 0.2% ninhydrin, 0.5% acetic acid, and 95% butan-1-ol. HPLC analysis was performed with a solvent containing 16 mm KH 2 PO 4 , 20% acetonitrile, and 0.04% phosphoric acid at a flow rate of 0.85 mLÆmin )1 at 40 °C, and eluates were monitored at 210 nm. Enzyme assays For the purification of the enzyme from P. putida, NMAADH activity was determined in a reaction mixture containing 12 mm phenylpyruvate, 240 mm methyl- amine ⁄ H 2 SO 4 (pH 10.0), and 8.0 mm NADPH in a final volume of 50 lLat37°C. The N-methylphenylalanine formed was analyzed by HPLC as described above. Other NMAADH assays were carried out by measuring the decrease in the amount of NADPH at 340 nm with an MPS-2000 spectrophotometer (Shimadzu, Kyoto, Japan) at 30 °C. A standard reaction mixture contained 10 mm phenylpyruvate, 60 mm methylamine ⁄ H 2 SO 4 (pH 10.0), 0.2 mm NADPH, and the enzyme in a final volume of 1 mL. Cloning and expression of NMAADH in E. coli PCR primers were designed based on the nucleotide sequence of ORF PP3591 located on the 4 080 935– 4 081 933-bp region of the P. putida KT2440 genome [37]. A 1-kb DNA fragment containing the NMAADH gene was amplified from the genomic DNA of another P. putida strain ATCC12633 by PCR with a Perkin–Elmer Thermal Cycler 480 (Wellesley, MA, USA) in a 50-lL reaction mix- ture containing 1· LA Taq buffer (Takara Shuzo), 2.5 mm MgCl 2 , 0.4 mm dNTP, 0.2 mm each primer (5¢-GGAAT TCCATATGTCCGCACCTTCCACCAGCACCG-3¢ and 5¢-GGGAAGCTTTCAGCCAAGCAGCTCTTTCAGG-3¢), 2.5 U LA Taq DNA polymerase, and 115 ng genomic DNA from P. putida ATCC12633: preincubation at 94 °C for 1 min and then 30 cycles between 98 °C for 20 s and 68 °C for 3 min and finally at 72 °C for 10 min. The PCR product was digested with NdeI and HindIII and ligated into pET21a(+) previously digested with the same restric- tion enzymes. The resultant plasmid, pDPKA, was intro- duced into E. coli BL21(DE3) to provide us with recombinant DpkA. Purification of recombinant NMAADH E. coli BL21(DE3) carrying pDPKA was cultivated in Luria–Bertani medium containing 100 lgÆL )1 ampicillin at 37 °C for 14 h. The culture was supplemented with 1 mm isopropyl b-d-thiogalactopyranoside and grown for 3 h. The wet cells (3.3 g) obtained by centrifugation were sus- pended in 28 mL of our standard buffer: a 20 mm Tris ⁄ HCl buffer (pH 7.0) containing 1 mm phenylmethanesulfonyl fluoride. The crude extract obtained by sonication was loa- ded on to a Green-Sepharose CL-4B column (100 mL) equilibrated with the standard buffer. The enzyme was elut- ed with a linear gradient of 0–1 m NaCl in the buffer. The enzyme fractions were pooled, concentrated with Centriprep YM-10 (Millipore, Bedford, MA, USA), and dialyzed against the buffer. The enzyme solution was applied to a DEAE-Toyopearl column (60 mL) equilibrated with the same buffer and eluted with a linear gradient of 0–0.35 m NaCl in the buffer. The enzyme fractions were collected, concentrated, and dialyzed against the standard buffer. The final preparation of the enzyme was stored at )80 °C until use without significant inactivation. Effect of various compounds on the activity The enzyme was incubated with various reagents in 20 mm Tris ⁄ HCl (pH 7.0) at 25 °C for 15 min. The remaining activity was determined as described above. Kinetic analysis Initial rates of the NMAADH reactions were measured with various concentrations of one substrate and fixed (and excess) concentrations of the other substrates. Data were fitted to the hyperbolic Michaelis–Menten equation, and kinetic parameters were calculated using nonlinear least-squares regression with kaleida graph software (Adelbeck Software, Reading, PA, USA) or IGOR Pro software (WaveMetrics, Inc., Lake Oswego, OR, USA). Eqn (1) describes a sequential mechanism: K a , K b , and K c represent the Michaelis constants for the NADPH (A), pyruvate (B), and methylamine (C), respectively; K ia and K ib are the dissociation constants for the enzyme–NADPH complex and the enzyme–pyruvate complex, respectively. Product inhibition studies were performed with various concentrations of either NADP + or N-methyl-l-alanine as one substrate and a fixed saturating concentration of the other. The data were fitted to Eqns (2), (3) and (4), which describe the competitive, uncompetitive, and noncompeti- tive inhibition patterns, respectively. P is the concentration of the product, K is is the inhibition constant from the slope H. Mihara et al. N-Methyl-L-amino acid dehydrogenase from P. putida FEBS Journal 272 (2005) 1117–1123 ª 2005 FEBS 1121 term, and K ii is the inhibition constant from the intercept term. v ¼ VABC=ðK ia K ib K c þ K ib K c A þ K ia K b C þ K c AB þ K b AC þ K a BC þ ABCÞð1Þ v ¼ VA=½K a ð1 þ P=K is ÞþAð2Þ v ¼ VA=½K a þ Að1 þ P=K ii Þ ð3Þ v ¼ VA=½K a ð1 þ P=K is ÞþAð1 þ P=K ii Þ ð4Þ Analytical size-exclusion chromatography The protein quaternary structure was analyzed by an A ¨ KTAexplorer system (Amersham Biosciences, Amersham, Buckinghamshire, UK) using a YMC-Pack Diol 200 col- umn (YMC Co, Ltd, Kyoto, Japan). The column was equilibrated and operated at a flow rate of 1.0 mLÆmin )1 with a 0.1 m potassium phosphate buffer (pH 7.0) contain- ing 0.2 m NaCl. The protein standards used were cyto- chrome c, myokinase, enolase, lactate dehydrogenase, and glutamate dehydrogenase from Oriental Yeast, Osaka, Japan. Other analytical methods The N-terminal amino-acid sequence of the enzyme was determined with an automated Shimadzu PPSQ10 protein sequencer (Kyoto, Japan). The nucleotide sequence of DNA was determined with an Applied Biosystems 370A DNA sequencer (Foster City, CA, USA). Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (B) 13125203 (to NE) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by Grant- in-Aid for Encouragement of Young Scientists 15780070 (to HM) from the Japan Society for the Pro- motion of Science, by the National Project on Protein Structural and Functional Analyses and by Grant-in- Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21st Century COE on Kyoto University Alliance for Chemistry). 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