1 Department of Bacteriology, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan
2 Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda, Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
3 First Department of Medicine, Yamanashi Medical University, Kofu, Yamanashi, 409-3898, Japan
Correspondence
Kumiko Nagata
kunagata{at}hyo-med.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Little study has been done on amino acids as respiratory substrates of H. pylori. H. pylori has genes encoding D-amino acid dehydrogenase (dadA), L-alanine dehydrogenase (ald) and L-serine deaminase (sdaB) (Tomb et al., 1997). These three enzymes produce pyruvate, which is the main respiratory substrate of H. pylori cells. In addition, H. pylori has a gene encoding proline dehydrogenase (putA), which is associated with the respiratory chain employing molecular oxygen as terminal electron acceptor in Escherichia coli and Salmonella typhimurium (Scarpulla & Soffer, 1978
; Menzel & Roth, 1981
). In this context, we have investigated the respiratory activity of intact whole H. pylori cells with alanine, serine and proline, and other amino acids of which both D- and L-isomers serve as respiratory substrates. In this report, we describe: (1) a high rate of utilization of L-serine and L-proline, followed by D-alanine and D-proline, as respiratory substrates; (2) an unusually high content of L-proline in H. pylori cells; and (3) the composition of free amino acids including D-isomers in gastric juice from patients infected with H. pylori and those from uninfected persons. These findings revealed an interesting relationship between some amino acids utilized as respiratory substrates and the composition of amino acids in gastric juice.
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METHODS |
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Respiration assay of H. pylori whole cells.
Respiration of whole cells (510x108 cells ml-1) was monitored polarographically with a Clark-type oxygen electrode (YSI Inc.) in a semi-closed vessel containing a medium of 10 mM HEPES buffer (pH 7·0) and 0·9 % NaCl at 37 °C as described previously (Nagata et al., 2001). The medium was bubbled with nitrogen gas to bring the oxygen concentration to 75 µM. Various kinds of amino acids and pyruvate with a final concentration of 10 mM and inhibitors such as rotenone and antimycin A were introduced into the oxygen electrode vessel with a syringe. Respiratory activity [oxygen uptake min-1 (mg protein)-1] was determined based on polarographic traces of the oxygen electrode as described previously (Nagata et al., 2001
).
Enzyme assay and protein determination.
Pyruvate production from amino acids was assayed as described previously (Nagata et al., 1988). Briefly, H. pylori cells were added to a reaction mixture containing 50 mM sodium phosphate buffer (pH 7·0), 10 mM NaN3 and 10 mM of amino acids. After 10 min incubation at 37 °C, 2,4-dinitrophenylhydrazine was added and the mixture kept at room temperature for 10 min. After addition of NaOH, hydrazone formation was measured by reading the A445 using a Beckman DU 640 spectrophotometer. The amount of pyruvate was calculated based on the A445 value of pure pyruvate as a standard.
2,6-Dichlorophenolindophenol (DCIP)-reducing activity was measured as follows. The cells were added to the reaction mixture containing 50 mM sodium phosphate buffer (pH 7·0), 10 mM of an amino acid and 0·5 mM DCIP. After 10 min incubation at 37 °C, the cells were removed by centrifugation at 10 000 g for 10 min and then the A600 of the supernatant was measured. An 600 of 21·6 mM-1 cm-1 was used.
Ammonia production was measured in the same reaction mixture as used for the assay of pyruvate production except without addition of NaN3. After incubation at 37 °C, the cells were removed by centrifugation at 10 000 g for 10 min, and the amounts of ammonia in the supernatant were measured by indophenol formation using the ammonia test reagent kit (Wako Pure Chemical Industry) as described previously (Nagata et al., 1993). The A630 was measured. Activities of pyruvate and ammonia production and of DCIP reduction were obtained by subtracting the activities without amino acids from those with amino acids.
The amount of protein of the whole H. pylori cells was determined by a modification of the Lowry procedure (Markwell et al., 1981).
Determination of free D- and L-isomers of amino acids in human gastric juice and H. pylori cells.
Gastric juice was collected from patients suspected of having gastroduodenal diseases. After pharyngeal anaesthesia with 1 % lidocaine hydrochloride, a disinfected endoscope was inserted into the stomach and gastric juice was collected through the aspiration channel of the endoscope. The status of H. pylori infection was determined from the results of culture and an immunological rapid urease test described previously (Sato et al., 2000). A patient was considered to be H. pylori-positive if the culture was positive. H. pylori-negative patients were defined as those with negative results for both tests. Gastroscopy was performed to evaluate the possibility of H. pylori infection. Informed consent was obtained from each patient prior to inclusion in the study.
To prepare the gastric juice sample, 30 % trichloroacetic acid solution was added to the juice to a final concentration of 5 % (w/v). Sample preparation from H. pylori cells has been described previously (Nagata et al., 1998). The supernatant of the trichloroacetic-acid-treated sample was passed through a Dowex 50Wx8 (H+ form) column and eluted with 4 M NH4OH after washing with distilled water to obtain purified free amino acids. The eluate was evaporated to dryness in vacuo in a centrifugal evaporator (Taitec) below 4 °C. The determination of amino acids of each enantiomer was performed as described previously (Nagata et al., 1992
). The free amino acids were treated with FDAA (Marfey's reagent, Pierce) (Marfey, 1984
) to form diastereomers of amino acids. The FDAA derivatives were separated on a Silica Gel 60 plate (Merck) by two-dimensional thin-layer chromatography. FDAA amino acids recovered from the plate were analysed by HPLC for the resolution of D- and L-enantiomers, using a reversed-phase column, Nova-Pak C18 (150x3·9 mm i.d., Waters), and a Tosoh or Jasco gradient HPLC system. The amounts of D- and L-enantiomers of the amino acids were calculated based on the peak areas of the elution patterns as obtained by a Chromato-Integrator (D-2500, Hitachi, Tokyo). Known amounts of authentic D- and L-enantiomers of each amino acid examined were added to the samples as internal controls, and subsequently hydrolysed and analysed as described.
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RESULTS |
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Pyruvate production from L-alanine added to whole cells of H. pylori was about 50 % of that from D-alanine (Table 2). Ammonia production was also about half that for D-alanine, showing a specific activity of 2·46 nmol min-1 (mg protein)-1. These results indicated that pyruvate production from L-alanine was due to L-alanine dehydrogenase, suggesting that this enzyme activity was weak compared to the D-amino acid dehydrogenase activity on D-alanine. These facts were consistent with the results that the respiratory activity on L-alanine was lower than that on D-alanine, as shown in Table 1
.
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DCIP-reducing activity of whole H. pylori cells with D- and L-isomers of alanine, serine and proline as substrate
D-amino acid dehydrogenase and L-alanine dehydrogenase show DCIP-reducing activity with concomitant production of pyruvate and ammonia (Olsiewski et al., 1980). We examined the DCIP-reducing activity of whole H. pylori cells using D- and L-isomers of alanine. Since H. pylori cells have strong activity of cytochrome c oxidase (Nagata et al., 1996
), the reduced DCIP is considered to be oxidized immediately. To prevent DCIP oxidation, we added various amounts of NaN3 to the reaction mixture used for the assay of DCIP reduction by D-alanine; the maximum DCIP reduction was obtained at 10 mM NaN3 (data not shown). In the presence of 10 mM NaN3, the specific activity of the DCIP reduction with D-alanine was close to that of pyruvate production with D-alanine (Table 2
).
L-alanine and D- and L-serine added to whole cells of H. pylori led to low DCIP-reducing activities (Table 2). On the other hand, high activities of DCIP reduction appeared with D- and L-proline, although the activities of pyruvate production from these amino acids were low (Table 2
).
Free amino acid composition of human gastric juice and H. pylori cells
Table 3 shows the contents of free D- and L-isomers of alanine, serine, proline, aspartate and glutamate in human gastric juices from H. pylori-infected and uninfected subjects. The content of L-proline in infected specimens was significantly higher than that in uninfected ones. The contents of D- and L-alanine and L-serine were also high. No significant difference was observed in amino acid composition, except for L-proline, between infected and uninfected specimens. As shown in Table 4
, H. pylori cells contained extremely large amounts of L-proline and considerable amounts of D- and L-alanine, L-serine and D-aspartate. The content of glutamate was low.
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DISCUSSION |
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In this report, we have presented evidence that H. pylori cells predominantly utilized D- and L-alanine and proline and also L-serine as respiratory substrates as well as pyruvate. The respiratory activities with these amino acids as substrate were inhibited by the respiratory inhibitors rotenone and antimycin A at low concentrations (Table 1), suggesting the respiratory activities to be coupled with ATP production. Although D-alanine as well as D-glutamate contributes to the synthesis of the cell wall of bacteria, other physiological functions and the metabolic pathways of these D-amino acids are equivocal in H. pylori cells. In the present study, we demonstrated that whole H. pylori cells utilized D-alanine but not D-glutamate as a respiratory substrate. As genome sequence analysis shows that H. pylori has the dadA gene, the reaction is considered to be the result of D-amino acid dehydrogenase function. Thus, oxygen uptake with D-alanine as substrate seems to have occurred via pyruvate, which is the main respiratory substrate in H. pylori cells (Mendz et al., 1994
). Considerably high activities of oxygen uptake and pyruvate production were found when L-serine was added to whole cells of H. pylori (Tables 1 and 2
). As H. pylori has a sdaA gene, this activity seems likely to be due to serine deaminase.
The presence of D-amino acid dehydrogenase has been reported in Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhimurium and Escherichia coli (Jones & Venables, 1983; Magor & Venables, 1987
; Olsiewski et al., 1980
; Tsukada et al., 1966
; Wild et al., 1974
). However, its metabolic function has not been clarified. D-Amino acid dehydrogenase and L-alanine dehydrogenase remove hydrogen from substrates of amino acids in the process of producing pyruvate and ammonia. Although the substances reduced by this hydrogen have not yet been identified in H. pylori cells, this hydrogen may be transferred to oxygen via the electron-transfer system leading to oxygen uptake by whole cells. However, it has not been clarified to what extent this hydrogen, besides pyruvate, participates in the oxygen uptake by D- and L-alanine in H. pylori cells.
In H. pylori, pyruvate is dehydrogenated by different dehydrogenase systems from those in the usual proteobacteria producing NADH, which is the major electron donor in the respiratory chain (Hughes et al., 1995; Kelly, 1998
). Two-step systems generating NADPH seem to be present in H. pylori; pyruvate may be oxidized by flavodoxin, and the reduced flavodoxin reduces NADP+. NADPH-menaquinone oxidoreductase oxidized NADPH, and the menaquinone may be oxidized by the cytochrome bc1 complex of the respiratory chain. Thus, the reducing system in pyruvate may be different from that in the hydrogen removed from D- and L-alanine.
Moreover, H. pylori showed marked activities of oxygen uptake with L- and D-proline as substrate, which were also inhibited by respiratory inhibitors (Table 2). E. coli, S. typhimurium and P. aeruginosa have the gene putA, whose product, PutA, shows L-proline dehydrogenase activity (Ling et al., 1994
; Vinod et al., 2002
). Recently, Satomura et al. (2002)
reported that the hyperthermophilic archaeon Pyrobaculum islandicum has a dye-linked D-proline dehydrogenase which is different from D-amino acid dehydrogenase. Both the L- and D-proline dehydrogenases have been reported to show DCIP reduction activity by the respective amino acids (Abrahamson et al., 1983
; Satomura et al., 2002
). In the present experiments, we demonstrated that H. pylori whole cells showed DCIP-reducing activity with D- and L-proline as substrate (Table 2
). H. pylori has the genes putP and putA, which encode proline permease and L-proline dehydrogenase, respectively (Tomb et al., 1997
). However, the gene corresponding to D-proline dehydrogenase has not been reported. From the genome sequence database, H. pylori dadA encoding D-amino acid dehydrogenase shows low DNA similarity (24 %) with Pb. islandicum dye-linked D-proline dehydrogenase (Satomura et al., 2002
). Since the substrate specificity of D-amino acid dehydrogenase may not be strict, this dehydrogenase may metabolize D-proline as a substrate for dcip-reducing activity as well as D-alanine. The hydrogen atoms removed from D- and L-proline by H. pylori whole cells may reduce a flavoprotein-like substance, and electrons produced concomitantly reduce oxygen via the electron-transport chain. Thus, different dehydrogenases and electron-transport pathways seem to participate in the oxidation of D- and L-isomers of alanine and proline. As shown in Table 2
, the inhibitory action of rotenone and antimycin A against respiratory activities varied considerably among respiratory substrates, being from 10 to 48 % of the control in the case of rotenone. These results may be due to the participation of different dehydrogenases and electron pathways depending on respiratory substrates.
Olson & Maier (2002) reported recently that H. pylori cells use molecular hydrogen as an energy source in mice. In the stomach of mice many kinds of anaerobic bacteria are present, in high density, and the molecular hydrogen derived from these anaerobic bacteria seems to be dominant. On the other hand, there are few bacteria in the human stomach, where amino acids are dominant owing to the degradation products of food, including L-serine derived from degradation of mucin. Since H. pylori resides in the lower layers of the gastric mucus, it is considered to use various amino acids diffused from the gastric juice as an energy source, although we cannot exclude the possibility that molecular hydrogen is also used by H. pylori in the human stomach as discussed by Olson & Maier (2002)
.
Previously, we reported the occurrence of free D-amino acids in various bacteria including H. pylori (Nagata et al., 1998). In the present study, we showed that of the amino acids contained in H. pylori cells, L-proline was present at the highest level, followed by D- and L-alanine, L-serine and D-aspartate (Table 4
). All these amino acids except D-aspartate were used predominantly as respiratory substrates by H. pylori whole cells as described above. We also analysed the L- and D-amino acid content of human gastric juice, which to the best of our knowledge is the first report of such measurement. We found considerable amounts of L- and D-isomers of alanine and proline, and of L-serine, in the gastric juice (Table 3
). Again, these amino acids coincided with the respiratory substrates used predominantly by H. pylori. Specimens from patients with H. pylori showed a significantly higher level of L-proline than those from uninfected subjects, although there was no significant difference in the contents of other free amino acids between the patients and the uninfected control group (Table 3
). There is the possibility that the free amino acids in gastric juice infected with H. pylori cells were derived from H. pylori cells since this organism contains a large amount of L-proline. H. pylori cells possess alanine racemase activity that converts L-alanine to D-alanine (unpublished observation). All these results, together with an apparently low Km value for the pyruvate-producing reaction from D-alanine, suggest that H. pylori cells utilize D-alanine, L-serine, and D- and L-proline as the main energy sources in their habitat of the mucous layer.
H. pylori cells possess high levels of urease. The hydrolysis of urea produces ammonia, which protects the organism from the highly acidic environment of the stomach. Not only urease but also enzymes that produce ammonia from amino acids, such as D-amino acid and L-alanine dehydrogenase, and L-serine deaminase, may contribute to protecting H. pylori cells from their acidic environment. Thus, the present study on the utilization of L-serine and D- and L-isomers of alanine in H. pylori cells offers important information on H. pylori metabolism, not only related to the energy source but also to protection measures against an acidic environment. These findings should be helpful for developing new anti-H. pylori therapies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Blaser, M. J. (1990). Helicobacter pylori and the pathogenesis of gastroduodenal inflammation. J Infect Dis 161, 626633.[Medline]
Burns, B. P. & Mendz, G. L. (2001). Metabolic transport. In Helicobacter pylori: Physiology and Genetics, pp. 207217. Edited by H. L. T. Mobley, G. L. Mendz & S. L. Hazell. Washington, DC: American Society for Microbiology.
Chang, H. T., Marcelli, S. W., Davison, A. A., Chalk, P. A., Poole, R. K. & Miles, R. J. (1995). Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori. FEMS Microbiol Lett 129, 3338.[CrossRef][Medline]
Doig, P., Jonge, B. L., Alm, R. A. & 10 other authors (1999). Helicobacter pylori physiology predicted from genomic comparison of two strains. Microbiol Mol Biol Rev 63, 675707.
Hughes, N. J., Chalk, P. A., Clayton, C. L. & Kelly, D. J. (1995). Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate : flavodoxin oxidoreductase from Helicobacter pylori. J Bacteriol 177, 39533959.[Abstract]
Jones, H. & Venables, W. A. (1983). Effects of solubilization on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli. FEBS Lett 151, 189192.[CrossRef][Medline]
Kelly, D. J. (1998). The physiology and metabolism of the human gastric pathogen Helicobacter pylori. Adv Microb Physiol 40, 136189.
Kelly, D. J., Hughes, N. J. & Poole, R. K. (2001). Microaerobic physiology: aerobic respiration, anaerobic respiration, and carbon dioxide metabolism. In Helicobacter pylori: Physiology and Genetics, pp. 113126. Edited by H. L. T. Mobley, G. L. Mendz & S. L. Hazell. Washington, DC: American Society for Microbiology.
Ling, M., Allen, S. W. & Wood, J. M. (1994). Sequence analysis identifies the proline dehydrogenase and 1pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein. J Mol Biol 243, 950956.[CrossRef][Medline]
Magor, A. M. & Venables, W. A. (1987). Solubilization, purification of D-amino acid dehydrogenase from Pseudomonas aeruginosa and effect of solubilization on its properties. Biochemie 69, 6369.[CrossRef][Medline]
Marfey, P. (1984). Determination of D-amino acids. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun 49, 591596.
Markwell, M. A. K., Haas, S. M., Tolbert, N. E. & Bieber, L. L. (1981). Protein determination in membrane and lipoprotein samples; manuals and automated procedures. Methods Enzymol 72, 19201931.
Mendz, G. L. & Hazell, S. L. (1993). Glucose phosphorylation in Helicobacter pylori. Arch Biochem Biophys 300, 522525.[CrossRef][Medline]
Mendz, G. L. & Hazell, S. L. (1995). Amimo acid utilization by Helicobacter pylori. Int J Biochem Cell Biol 27, 10851093.[CrossRef][Medline]
Mendz, G. L., Hazell, S. L. & Burns, B. P. (1993). Glucose utilization and lactate production by Helicobacter pylori. J Gen Microbiol 139, 30233028.[Medline]
Mendz, G. L., Hazell, S. L. & van Gorkom, L. (1994). Pyruvate metabolism in Helicobacter pylori. Arch Microbiol 162, 187192.[CrossRef][Medline]
Menzel, R. & Roth, J. (1981). Enzymatic properties of the purified put A protein from Salmonella typhimurium. J Biol Chem 256, 97629766.
Nagata, Y., Shimojo, T. & Akino, T. (1988). Two spectrophotometric assays for D-amino acid oxidase: the study of distribution patterns. Int J Biochem 20, 12351238.[Medline]
Nagata, K., Satoh, H., Iwahi, T., Shimoyama, T. & Tamura, T. (1993). Potent inhibitory action of the gastric proton pump inhibitor lansoprazole against urease activity of Helicobacter pylori: unique action selective for H. pylori cells. Antimicrob Agents Chemother 37, 769774.[Abstract]
Nagata, K., Tsukita, S., Tamura, T. & Sone, N. (1996). A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori. Microbiology 142, 17571763.[Abstract]
Nagata, Y., Fujiwara, T., Kawaguchi-Nagata, K., Fukumori, Y. & Yamanaka, T. (1998). Occurrence of peptidyl D-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes. Biochim Biophys Acta 1379, 7682.[Medline]
Nagata, Y., Yamamoto, K. & Shimojo, T. (1992). Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. J Chromatogr 575, 147152.[Medline]
Nagata, K., Sone, N. & Tamura, T. (2001). Inhibitory activities of lansoprazole against respiration in Helicobacter pylori. Antimicrob Agents Chemother 45, 15221527.
Olsiewski. , Kaczorowski, G. J. & Walsh, C. (1980). Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B. J Biol Chem 255, 44874494.
Olson, J. W. & Maier, R. J. (2002). Molecular hydrogen as an energy source for Helicobacter pylori. Science 298, 17881790.
Parsonnet, J., Friedman, G. D. Vandersteen D. P., Chang, Y., Vogelman, J. H., Orentreich, N. & Sibley, R. K. (1991). Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 325, 11271131.[Abstract]
Parsonnet, J., Hansen, S., Rodriguez, L., Gelb, A. B., Warnke, R. K., Jellum, E., Orentrich, N., Vogelman, J. H. & Friedman, G. D. (1994). Helicobacter pylori infection and gastric lymphoma. N Engl J Med 330, 12671271.
Rabeneck, L. & Ransohoff, D. F. (1991). Is Helicobacter pylori a cause of duodenal ulcer? A methodologic critique of current evidence. Am J Med 91, 566572.[Medline]
Reynolds, D. J. & Penn, C. W. (1994). Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140, 26492656.[Abstract]
Sato, T., Fujino, M. A., Koijima, Y., Kitahara, F., Morozumi, A. Nagata K., Nakamura, M. & Hosaka, H. (2000). Evaluation of immunological rapid urease testing for detection of Helicobacter pylori. Eur J Microbiol Infect Dis 19, 438442.[CrossRef]
Satomura, T., Kawakami, R., Sakuraba, H. & Ohshima, T. (2002). Dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase. J Biol Chem 277, 1286112867.
Scarpulla, R. C. & Soffer, R. J. (1978). Membrane-bound proline dehydrogenase from Escherichia coli. Solubilization, purification, and characterization. J Biol Chem 253, 59976001.[Medline]
Stark, R. M., Suleiman, M. S., Hassan, I. J., Greenman, J. & Millar, M. R. (1997). Amino acid utilization and deamination of glutamine and asparagine by Helicobacter pylori. J Med Microbiol 46, 793800.[Abstract]
Tomb, J. F., White, O., Kerlavage, A. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539547.[CrossRef][Medline]
Tsukada, K. (1966). D-Amino acid dehydrogenase of Pseudomonas fluorescens. J Biol Chem 241, 45224528.
Vinod, M. P., Bellur, P. & Becker, D. F. (2002). Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli. Biochemistry 41, 65256532.[CrossRef][Medline]
Wild, J., Walczak, W., Krajewaka-Crynkiewicz, K. & Klopotowski, T. (1974). D-Amino acid dehydrogenase: the enzyme of the first step of D-histidine and D-methionine racemization in Salmonella typhimurium. Mol Gen Genet 128, 131146.[Medline]
Yonaha, K., Misono, H., Yamamoto, T. & Soda, K. (1975). D-Amino acid aminotransferase of Bacillus sphaericus. J Biol Chem 250, 69836989.[Abstract]
Received 23 December 2002;
revised 10 April 2003;
accepted 22 April 2003.
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