Abstract
l-Lysine dehydrogenase, which catalyzes the oxidative deamination of l-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ1-piperideine-6-carboxylate, indicating that the enzyme is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM l-lysine. The enzyme was relatively selective for l-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for l-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.
l-Lysine dehydrogenases catalyze the oxidative deamination of l-lysine in the presence of NAD; so far, two types of l-lysine dehydrogenases have been identified (Fig. 1). The first type is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18), which catalyzes the oxidative deamination of the ɛ-amino group of l-lysine to form l-2-aminoadipate-6-semialdehyde, which in turn nonenzymatically cyclizes to form Δ1-piperideine-6-carboxylate (17). The second type of l-lysine dehydrogenases is l-lysine 2-dehydrogenase (EC 1.4.1.15), which catalyzes the oxidative deamination of the α-amino group of l-lysine. LysDH was first found in Agrobacterium tumefaciens, in which it plays a key role in l-lysine metabolism (16). Since then, this enzyme has been purified, characterized (8, 15, 18), and utilized for l-lysine determination (4, 9). In addition to the A. tumefaciens LysDH, the enzyme from the yeast Candida albicans has been partially characterized (7). In contrast, there has been only one report of a human liver l-lysine 2-dehydrogenase catalyzing the deamination of the α-amino group (2). In this case, however, the reaction product has yet to be identified; indeed, it is still not completely clear which amino group the enzyme cleaves.
FIG. 1.
Reaction scheme for two l-lysine dehydrogenases.
To date, about 17 different amino acid dehydrogenases have been identified in various organisms (21, 22). Some of these enzymes are useful for enzymatic analysis of amino acids, oxo acids, and special enzymes and for the stereospecific synthesis of l-amino acids and their analogues (10, 21, 22). In contrast to the abundant information available about glutamate, alanine, leucine, and phenylalanine dehydrogenases (21), information about the structure and function of lysine dehydrogenase is rather limited due to the instability of the A. tumefaciens enzyme. In particular, there have been no reports so far on the primary structure of lysine dehydrogenases. In the present study, therefore, we screened thermophilic and hyperthermophilic microorganisms for a more stable form of l-lysine dehydrogenase and found one such enzyme in a moderately thermophilic bacterium, Geobacillus stearothermophilus, which was isolated from a Japanese hot spring. Here we describe purification and characterization of the enzyme protein, as well as cloning, sequencing, and recombinant expression of its gene.
MATERIALS AND METHODS
Materials.
SuperQ-TOYOPEARL 650M was purchased from Tohso (Tokyo, Japan). Gigapite and Cellulofine GC-700-m were obtained from Seikagaku Kogyo (Tokyo, Japan). Hybond-N+ membranes, a Probe QuantTM G-50 Micro column, and [γ-32P]ATP were obtained from Amersham Pharmacia Biotech. Plasmid pUC18 DNA and MEGALABEL were obtained from Takara Biochemicals (Kyoto, Japan). Salmon sperm DNA was purchased from Sigma Chemical Co. Restriction endonucleases, DNA-modifying enzymes, molecular mass markers for electrophoresis, and other chemicals were obtained commercially. Red-Sepharose CL-4B was prepared as described previously (20).
Microorganisms and growth conditions.
A thermophilic bacterium producing lysine dehydrogenase was isolated from the mud of an Onikobe hot spring in Miyagi prefecture, Japan. Based on the nucleotide sequence of its 16S rRNA and its physiological properties, this organism was identified as G. stearothermophilus strain UTB 1103 (unpublished data), and it was aerobically cultivated at 60°C for about 12 h in medium (pH 7.0) containing 1% peptone, 0.5% yeast extract, 0.05% KH2PO4, 0.1% K2HPO4, and 0.02% MgSO4 · 7H2O. Other thermophilic and hyperthermophilic microorganisms were obtained mainly from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and the Japan Collection of Microorganisms (Wako, Japan). A. tumefaciens ICR 1600 was provided by T. Yoshimura of the Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu, Japan, and was grown in 500 ml of medium (pH 7.2) containing 0.5% l-lysine, 0.5% peptone, 0.2% NaCl, 0.2% K2HPO4, 0.2% KH2PO4, 0.01% MgSO4 · 7H2O, and 0.01% yeast extract as previously described (16). Recombinant strains of Escherichia coli JM 109 were cultivated in Luria-Bertani medium (1% tryptone, 0.5% yeast extract, 1% NaCl; pH 7.2) supplemented with 50 μg of ampicillin per ml and 100 μg of isopropyl-β-d-thiogalactopyranoside per ml (if necessary) at 37°C.
Enzyme and protein concentration assays.
LysDH activity was spectrophotometrically assayed by using a Shimadzu UV-160A recording spectrophotometer equipped with a thermostat. The standard reaction mixture (final volume, 1.0 ml) contained 100 mM glycine-KOH buffer (pH 10.0), 1.25 mM NAD, 10 mM l-lysine, and the enzyme. After the reaction mixture was initially incubated without l-lysine at 50°C for about 3 min in a cuvette with a 1-cm light path, the reaction was started by addition of l-lysine, and the increase in the absorbance at 340 nm was monitored for 2 min. The increase in the absorbance at 340 nm measured with a reaction mixture without l-lysine was subtracted from the total signal as the blank. With the amount of enzyme used, the absorbance increased linearly for at least 2 min. One unit of enzyme was defined as the amount that catalyzed the formation of 1 μmol of NADH under the standard assay conditions. An absorption coefficient at 340 nm (ɛ) of 6.22 mM−1 cm−1 was used for NAD(P)H. The protein concentration was measured by the method of Bradford (1); bovine serum albumin was used as the standard.
Purification of LysDH from G. stearothermophilus.
All purification procedures were carried out at a temperature below 4°C. The cells harvested from 15 liters of culture (ca. 24 g, wet weight) were used as the starting material for the purification of LysDH. Unless indicated otherwise, 10 mM potassium phosphate buffer (pH 7.0) containing 10% glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol was used as the standard buffer throughout the purification procedure in this study.
(i) Preparation of crude extract.
To prepare a crude extract, the cells were washed twice with the standard buffer and suspended in this buffer, after which they were lysed with an ultrasonic disrupter (TOMY UD-200) and centrifuged at 11,400 × g for 10 min. The resultant supernatant was used as the crude extract.
(ii) SuperQ-TOYOPEARL 650M column chromatography.
The crude extract was applied to a SuperQ-TOYOPEARL 650M column (diameter, 5 cm; length, 15 cm) equilibrated with the standard buffer. The column was then washed with the buffer and eluted with a linear gradient of 0 to 0.6 M NaCl in buffer. LysDH was eluted at an NaCl concentration of about 0.15 M, and the active fractions were combined and dialyzed against the standard buffer.
(iii) Red-Sepharose CL-4B column chromatography.
The dialysate was applied to a Red-Sepharose CL-4B column (diameter, 2.8 cm; length, 10 cm) equilibrated with the standard buffer, after which the column was washed with the buffer and the enzyme was eluted with a linear gradient of 0 to 1.5 M NaCl in the buffer. The enzyme was eluted at an NaCl concentration of about 0.5 M, and the active fractions were combined and dialyzed against the standard buffer.
(iv) Gigapite column chromatography.
The second dialysate was applied to a Gigapite column (diameter, 1.3 cm; length, 10 cm) equilibrated with the standard buffer. When the column was washed with the buffer, the enzyme passed through it without adsorption. The active fractions were combined and dialyzed against the standard buffer containing 0.1 M NaCl.
(v) Cellulofine GC-700-m gel filtration column chromatography.
The third dialysate was applied to a Cellulofine GC-700-m column (diameter, 2.6; length, 63 cm) equilibrated with the standard buffer containing 0.1 M NaCl, and the active fractions were combined and dialyzed against the same buffer. The resultant enzyme solution was used as the final LysDH preparation.
PAGE.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out as described by Laemmli (12) by using 12.5% acrylamide and 0.1% SDS in a discontinuous Tris-glycine buffer system. Maltose-binding protein-β-galactoside (molecular mass, 175 kDa), maltose-binding protein-paramyosin (83 kDa), glutamate dehydrogenase (62 kDa), aldolase (47.5 kDa), triose phosphate isomerase (32.5 kDa), β-lactoglobulin A (25 kDa), lysozyme (16.5 kDa), and aprotinin (6.5 kDa) were used as molecular mass standards (New England Biolabs). The protein sample was boiled for 5 min in 10 mM Tris-HCl buffer (pH 7.0) containing 1% SDS and 1% 2-mercaptoethanol. Protein bands were visualized by staining with Coomassie brilliant blue R-250.
Native gradient PAGE for molecular mass determination was carried out by using the method of Manabe et al. (14), with some modifications; the gel used was a 2 to 15% polyacrylamide linear gradient slab gel, which was electrophoresed with a running buffer containing 90 mM Tris-HCl, 80 mM boric acid, and 2.5 mM EDTA (pH 8.4) at 4°C. The standard proteins (Bio-Rad) used to construct a calibration curve for the native enzyme included bovine thyroglobulin (molecular mass, 670 kDa), horse spleen ferritin (443 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin (66 kDa), and tripsin inhibitor (20 kDa). The resolved bands were stained with Coomassie brilliant blue R-250.
Identification of the product of oxidative deamination of l-lysine.
To confirm that the enzyme isolated from G. stearothermophilus was in fact LysDH (EC 1.4.1.18), its reaction product was identified by thin-layer chromatography (TLC). The postulated product, Δ1-piperideine-6-carboxylate, is not commercially available, so we prepared it using A. tumefaciens LysDH (17). Before use, A. tumefaciens LysDH was partially purified by ammonium sulfate fractionation (40 to 60% saturation) and DEAE-cellulose column chromatography as described previously (17). To identify the product, 50 μl of a reaction mixture containing 20 mM l-lysine, 5 mM NAD, and 50 mM NaHCO3-NaOH (pH 9.5) was incubated overnight at 30°C with the A. tumefaciens enzyme, or 50 μl of a reaction mixture containing 20 mM l-lysine, 5 mM NAD, and 50 mM NaHCO3-NaOH (pH 9.5) was incubated overnight at 50°C with the G. stearothermophilus enzyme. Each reaction was stopped by addition of 5 μl of 30% perchloric acid (HClO4), the mixture was centrifuged at 13,000 × g for 10 min, and 6 μl of 2 M K2CO3 was added to the supernatant, which was then centrifuged at 15,000 × g for 10 min. An aliquot (5 μl) of the resultant supernatant was then applied to silica gel TLC plates (Whatman, Kent, England) and developed at room temperature by using either a phenol solution containing 3% ammonia or a mixture of n-butanol, acetic acid, and water (4:2:1). The Δ1-piperideine-6-carboxylate formed and the remaining substrate (l-lysine) were separated and detected with ninhydrin (0.2% [wt/vol] in acetone). Under these conditions, l-lysine migrated with Rf values of 0.11 and 0.22 in phenol-ammonia and in n-butanol-acetic acid-water, respectively, whereas Δ1-piperideine-6-carboxylate migrated with Rf values of 0.27 and 0.33, respectively.
Analysis of the N-terminal amino acid sequence of LysDH.
Samples (about 1 nmol) of purified enzyme were subjected to SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. The N-terminal amino acid sequence was then analyzed by automated Edman degradation by using a Shimadzu model PPSQ-10 gas phase sequencer.
DNA probe preparation.
To screen for the LysDH gene, a probe consisting of a mixture of oligonucleotides containing nine inosines (44-mer, 64 kinds;e.g., 5′-ATGAARGTIYTIGTIYTIGGIGCIGGIYIATGGGIAARGARGC-3′)(probe A) was synthesized based on the previously determined N-terminal amino acid sequence of the enzyme (MKVLVLGAGLMGKEAARDLVQSQDV). Probe A was then labeled with [γ-32P]ATP by using T4 polynucleotide kinase with MEGALABEL and used as a specific probe for colony and Southern hybridizations.
Construction of a genomic library and screening for positive clones.
Genomic DNA from G. stearothermophilus UTB 1103 was prepared as follows. Cells (ca. 2 g, wet weight) were suspended in 3 ml of buffer (50 mM Tris-HCl [pH 8.0], 50 mM EDTA, 10 mg of lysozyme per ml, 5 mg of pronase per ml, 160 mg of RNase A per ml) and incubated at 37°C for 4 h with gentle shaking. This was followed by addition of 2 ml of a STEP solution (50 mM Tris-HCl [pH 8.0], 400 mM EDTA, 0.5% SDS, 6 mg of proteinase K per ml) and incubation at 50°C for 1 h with gentle shaking. The proteins in the mixture were then extracted several times with phenol-chloroform, and the genomic DNA was precipitated with 0.7 volume of isopropanol. About 2.9 mg of DNA was obtained from 2 g of cells.
To screen for the LysDH gene, samples of genomic DNA prepared from G. stearothermophilus UTB 1103 were digested with SphI. After this, plasmid vector pUC18 was digested with SphI, treated with alkaline phosphatase, and ligated with the genomic DNA SphI fragments by using T4 ligase. The resultant vector was used to transform E. coli JM 109 cells, which enabled them to serve as a genomic library for screening the LysDH gene. Colony hybridization was carried out as previously described (19). Recombinant colonies from the genomic library were transferred to nylon membranes (Hybond-N+) and fixed, after which prehybridization and hybridization were carried out according to the manufacturers' instructions. One positive clone was detected by using a BAS-1500 system (Fuji Film, Tokyo, Japan) and was confirmed by Southern hybridization.
DNA sequencing and amino acid sequence analysis.
Nucleotide sequencing was carried out by the dideoxy chain termination method (24) with an automated 377A DNA sequencer (Applied Biosystems), after which the nucleotide sequence was analyzed by using the GENETYX gene analysis software (Software Development, Tokyo, Japan).
Purification of LysDH from recombinant E. coli expressing the enzyme activity.
E. coli strain JM 109 cells were transformed with plasmid pNN53, after which the transformants were plated on Luria-Bertani medium supplemented with ampicillin (50 μg/ml) and isopropyl-β-d-thiogalactopyranoside (100 μg/ml) and grown aerobically at 37°C for about 12 h. About 8 g (wet weight) of cells was harvested from the 2-liter culture. Unless indicated otherwise, all procedures described below were carried out at room temperature (ca. 25°C).
(i) Preparation of crude extract.
The transformed cells were suspended in the standard buffer, disrupted by sonication at 4°C, and centrifuged at 15,000 × g for 15 min. The resultant supernatant was used as the crude extract.
(ii) First Red-Sepharose column chromatography.
The crude extract was applied to a Red-Sepharose CL-4B column (diameter, 5 cm; length, 11 cm) previously equilibrated with the standard buffer. After the column was washed with the same buffer, the enzyme was eluted with a linear gradient of 0 to 1.5 M NaCl in the buffer. The active fractions were pooled and dialyzed against the standard buffer.
(iii) Second Red-Sepharose column chromatography.
The dialysate was applied to a second Red-Sepharose CL-4B (diameter, 5 cm; length, 5 cm) previously equilibrated with the standard buffer supplemented with 1 mM l-lysine, and then it was eluted with a linear gradient of 0 to 1 mM NAD in buffer containing 1 mM l-lysine. The active fractions were pooled and dialyzed against the standard buffer, and the resultant dialysate was used as the final purified recombinant LysDH preparation.
Nucleotide sequence accession number.
The nucleotide sequence determined in this study has been deposited in the DDBJ, GenBank, and EMBL data banks under accession number AB052732.
RESULTS
Distribution of LysDH in thermophilic bacteria.
To find organisms that produce thermostable NAD-dependent lysine dehydrogenase, we screened enzymes which catalyze the oxidative deamination of l-lysine in the presence of NAD in several strains of thermophilic bacteria and hyperthermophilic archaea from culture collections, including Pyrococcus furiosus DSM 3638, Pyrococcus horikoshii OT-3, Thermococcus litoralis DSM 5473, Thermococcus profundus DSM 9503, Thermococcus peptonophilus DSM 10343, Pyrobaculum islandicum DSM 4184, Aeropyrum pernix JCM 9820, Sulfolobus tokodaii JCM 10545, G. stearothermophilius DSM 297, DSM 456, DSM 458, DSM 494, IFO 12550, and UTB 1103, Bacillus sphaericus DSM 461 and DSM 462, and Thermus sp. strain UTB 1104. We found LysDH activity in only G. stearothermophilus UTB 1103 (specific activity, around 2.05 mU/mg); the other strains did not exhibit any LysDH activity. Therefore, G. stearothermophilus UTB 1103 was used for enzyme purification.
Purification and properties of LysDH.
LysDH was initially purified to homogeneity from a crude extract of G. stearothermophilus UTB 1103 by using four successive chromatography steps (Table 1). The Super Q-TOYOPEARL 650M step was notable in that it increased the total activity of enzyme significantly, most likely as a result of removing endogenous inhibitors. Ultimately, the enzyme was purified about 3,040-fold, with an overall yield of about 63%. By using native gradient PAGE, the molecular mass of the isolated enzyme was estimated to be about 260 kDa. When SDS-PAGE was used, the molecular mass of the subunit was determined to be about 43 kDa and the enzyme was resolved as a single band, indicating that the intact protein is a homohexamer (Fig. 2). The N-terminal amino acid sequence of the purified enzyme was determined to be MKVLVLGAGLMGKEAARDLVQSQDV.
TABLE 1.
Purification of LysDH from G. stearothermophilus and recombinant E. coli LysDH
| Step | Total protein (mg) | Total activity (u) | Sp act (u/mg) | Yield (%) |
|---|---|---|---|---|
| G. stearothermophilus LysDH | ||||
| Crude extract | 2,520 | 5.18 | 0.00205 | 100 |
| SuperQ-TOYOPEARL 650M | 53.4 | 21.9 | 0.369 | 424 |
| Red-Sepharose CL-4B | 3.32 | 7.21 | 2.12 | 139 |
| Gigapite | 1.04 | 4.66 | 4.48 | 90.0 |
| Cellulofine GC-700-m | 0.521 | 3.25 | 6.24 | 62.7 |
| Recombinant LysDH | ||||
| Crude extract | 598 | 995 | 1.67 | 100 |
| First Red-Sepharose CL-4B | 186 | 664 | 3.57 | 67 |
| Second Red-Sepharose CL-4B | 75.5 | 590 | 7.81 | 59 |
FIG. 2.
SDS-PAGE of the purified enzyme. After LysDH from G. stearothermophilus was purified as described in Materials and Methods, the proteins were fractionated by SDS-PAGE, and the gel was stained with Coomassie brilliant blue R-250.
TLC revealed that the reaction product resulting from oxidative deamination of l-lysine by the enzyme from G. stearothermophilus LysDH was identical to that of A. tumefaciens LysDH (Δ1-piperideine-6-carboxylate). Thus, the G. stearothermophilus lysine dehydrogenase belongs to the family of LysDH (EC 1.4.1.18), which catalyzes the oxidative deamination of the ɛ-amino group of l-lysine.
Effects of pH, temperature, and various chemicals on enzyme activity and stability.
The effect of pH on the oxidative deamination of l-lysine was determined at various pHs at 50°C, and the optimum pH was about 10.0 (Fig. 3). When the temperature dependence of the catalytic activity at pH 10.0 was examined, the maximum activity was found to occur at temperatures around 70°C (data not shown). Moreover, when the enzyme was incubated for 10 min at various temperatures in the standard buffer system at pH 7.2, full activity was retained at temperatures up to 60°C; however, the activity was markedly reduced at 65°C (Fig. 4). The thermostability of LysDH was improved by addition of 5 mM l-lysine, in the presence of which the enzyme retained activity at temperatures up to 65°C. When the effect of pH on the stability of the enzyme was examined by incubation at 50°C for 30 min, the enzyme was most stable at pH 6.0 to 9.0 (data not shown). At low temperatures (e.g., 4°C), LysDH could be stored for more than 3 months without a loss of activity.
FIG. 3.
Effect of pH on G. stearothermophilus LysDH activity. Enzyme activity was measured at various pHs at 50°C by using 100 mM buffers, including K2HPO4-KH2PO4 (pH 7.0 to 7.5) (⧫), Tris-HCl (pH 7.5 to 8.5) (▪), and Gly-KOH (pH 8.5 to 11.0) (▴). Enzyme activity values are expressed as percentages relative to that determined in one experiment in which 0.032 U of enzyme was used.
FIG. 4.
Effect of temperature on the stability of G. stearothermophilus LysDH. The enzyme was incubated for 10 min at different temperatures in 50 mM K2HPO4-KH2PO4 buffer (pH 7.2) with (○) or without (•) 5 mM l-lysine. The remaining activity was measured under the standard assay conditions. The amount of enzyme was determined to be about 0.032 U in 100% points.
Examination of the effects of several chemicals on the activity of LysDH (in 10 mM potassium phosphate buffer, pH 7.0) showed that the enzyme was inhibited by 1 mM p-chloromercuribenzoic acid (remaining activity, 67%), 1 mM Pb(CH3COO)2 (63%), and 1 mM HgCl2 (15%) but was not affected by EDTA, BaCl2, MnCl2, MgCl2, NiCl2, CaCl2, CoCl2, ZnCl2, AlCl3, FeCl2, CuCl2, SrCl2, or NaN3 (each at a concentration of 1 mM).
Substrate and coenzyme specificities.
Examination of the substrate specificity of the enzyme revealed that l-lysine is the preferred substrate for oxidative deamination in the presence of NAD, although the enzyme showed low activity with S-(β-aminoethyl) l-cysteine (the relative activity was 11% of the activity with l-lysine). The following amino acids (at a concentration of 10 mM, unless indicated otherwise) were found to be inert (i.e., they supported no catalytic activity at all): d-lysine, l-ornithine, l-phenylalanine, l-arginine, l-tyrosine (3 mM), l-histidine, l-proline, l-aspartate, l-asparagine, l-glutamic acid, l-serine, l-threonine, l-methionine, l-leucine, l-isoleucine, l-valine, glycine, l-alanine, l-cysteine, and l-tryptophan.
Both NAD and NADP were able to serve as the coenzyme for LysDH, although the reaction rate with NADP (2.5 mM) was only 22% of that with NAD (2.5 mM) when l-lysine was used as the substrate. Analysis of the initial velocity of the oxidative deamination of l-lysine in the presence of NAD yielded typical Michaelis-Menten kinetics. As determined by Lineweaver-Burk plots, the apparent Km values at 50°C in Gly-KOH buffer (pH 10.0) for l-lysine, NAD, and NADP were 0.73, 0.088, and 0.48 mM, respectively.
Gene cloning and sequencing.
To clone the LysDH gene, an SphI genomic library of G. stearothermophilus, consisting of about 20,000 transformants, was screened by using a 32P-labeled oligonucleotide mixture as the probe. After the recombinant plasmids were screened by Southern hybridization, one positive clone, pNN51, which harbored a 3.9-kbp SphI fragment, was selected for further analysis. Sequencing of the fragment revealed a single 1,155-bp open reading frame that encoded 385 amino acids, and the deduced N-terminal amino acid sequence was identical to the sequence obtained by protein sequencing. The translation product had a molecular mass of 42,239 Da.
Amino acid sequence alignment.
When similarities to the amino acid sequence of G. stearothermophilus LysDH were sought among the sequences in the GenBank databases by using the BLAST server, some similarity was found to the Oceanobacillus iheyensis HTE831 hypothetical protein (65% identity) (13, 25); somewhat lower levels of similarity were found to a P. horikoshii enzyme (33% identity) (11), a Thermoplasma acidophilum enzyme (31% identity) (23), and A. tumefaciens strain C58 dehydrogenase (30% identity) (6, 27).
Gene expression in E. coli, purification of the recombinant enzyme, and primary properties of the recombinant enzyme.
A 2.1-kbp NotI-SphI fragment containing the G. stearothermophilus LysDH gene from pNN51 was subcloned into pUC18, yielding pNN53, which was then used to transform E. coli JM 109 cells. The crude extract from the recombinant cells showed a high level of LysDH activity (Table 1), and by using two successive dye affinity chromatography steps, 75.5 mg of the purified enzyme was obtained from the extract prepared from 2 liters of culture. Automated Edman degradation showed that the 25 N-terminal amino acids of the recombinant LysDH were identical to those of G. stearothermophilus UTB 1103 LysDH, while SDS-PAGE showed that the subunit molecular mass was identical to that of the wild-type enzyme (43 kDa). The optimum pH for activity of the recombinant enzyme was in the range from pH 10 to 10.6, and the enzyme was stable at pHs ranging from 6.5 to 10.0 when it was incubated for 30 min at 50°C.
Like wild-type G. stearothermophilus UTB 1103 LysDH, the catalytic activity of the recombinant enzyme was stable for 10 min at temperatures up to 60 and 65°C in the absence and presence of 5 mM l-lysine, respectively, and the optimum temperature for activity was around 70°C. The substrate and coenzyme specificities also showed some similarity to those of the wild-type enzyme; the apparent Km values for l-lysine, NAD, and NADP for the recombinant enzyme at 50°C in Gly-KOH buffer (pH 10.0) were estimated to be 0.44, 0.061, and 0.40 mM, respectively.
DISCUSSION
In the present study, we screened a large number of thermophilic and hyperthermophilic bacteria and archaea with the aim of obtaining a more stable and useful form of l-lysine dehydrogenase. We found a form of LysDH in the thermophilic bacterium G. stearothermophilus UTB 1103, which was isolated from a hot spring. It is noteworthy that while Misono and Nagasaki observed lysine dehydrogenase activity in many mesophiles, they did not find it in thermophiles (17), which makes this the first report of the presence of LysDH in a thermophilic bacterium.
As we expected, LysDH purified from G. stearothermophilus was much more stable than its counterpart from the mesophilic organism A. tumefaciens. Whereas the A. tumefaciens enzyme loses activity when it is incubated at temperatures above 37°C for 10 min (15), the G. stearothermophilus enzyme remains fully active at temperatures up to 65°C. In addition, G. stearothermophilus LysDH is highly stable over a wide range of pHs and can be stored for long periods at low temperatures. This is noteworthy because the high stability of G. stearothermophilus LysDH under a variety of conditions makes it comparatively simple to prepare the highly purified preparations needed for bioprocesses. For instance, the G. stearothermophilus enzyme could be used for production of l-pipecolic acid (5) and 2-aminoadipic acid (3) from l-lysine via Δ1-piperideine-6-carboxylate. Still, A. tumefaciens LysDH has been utilized for enzymatic analysis of l-lysine (9) and as an l-lysine biosensor (4). Like the A. tumefaciens enzyme, G. stearothermophilus LysDH is highly specific for l-lysine, which is advantageous for use in a biosensor. On the other hand, unlike the A. tumefaciens enzyme, G. stearothermophilus LysDH exhibits low specificity for NAD and NADP. NADP is totally inactive as a coenzyme with A. tumefaciens LysDH (15), but it has about 22% of the activity of NAD with the G. stearothermophilus enzyme, which should also add to the versatility and applicability of this enzyme (26).
When the gene for G. stearothermophilus LysDH was cloned and sequenced, the deduced amino acid sequence (385 amino acids) showed that the N-terminal region contains a typical NAD-binding motif (GXGXXGX21D, βαβ-fold) (26). This is the first information available on the primary structure of LysDH, and BLAST searches of the GenBank databases showed some homology to the O. iheyensis HTE831 hypothetical protein (OB0862; level of homology, 65%), a P. horikoshii hypothetical protein (PH1688; level of homology, 33%), a T. acidophilum hypothetical protein (TA0681; level of homology, 31%), and an A. tumefaciens protein (AGR_C_1727p; level of homology, 30%). Finally, we were able to overproduce thermostable LysDH in E. coli cells. This is the first example of overproduction of LysDH, and the specific activity of the crude E. coli extract was about 800-fold higher than the activity of the G. stearothermophilus extract (Table 1). The high level of enzyme expression should enable larger-scale preparation of the purified enzyme, which in turn should enable more detailed investigation of the relationship between the structure and function of LysDH and the applicability of this enzyme to various bioprocesses.
Acknowledgments
We sincerely appreciate the technical support of Ryushi Kawakami, Kyoko Honda, Yoshinori Takamatsu, Hiromi Ishikawa, and Yuka Sasaki. We thank T. Yoshimura, Institute for Chemical Research, Kyoto University, for his kind gift of A. tumefaciens strain ICR 1600.
M. Heydari was supported by a government scholarship from the Ministry of Education, Science, Sports and Culture of Japan.
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