Abstract
A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked d-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked d-amino acid dehydrogenase that retained more than 80 % of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, d-phenylalanine was the most preferable substrate among the d-amino acids tested. Immediately upstream of the dye-linked d-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy-l-proline in R. marinus. This is the first description of a dye-linked d-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy-l-proline catabolism.
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Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientific Research (B) to Takenori Satomura from the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research (A) to Toshihisa Ohshima.
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Satomura, T., Ishikura, M., Koyanagi, T. et al. Dye-linked d-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-l-proline catabolism. Appl Microbiol Biotechnol 99, 4265–4275 (2015). https://doi.org/10.1007/s00253-014-6263-9
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DOI: https://doi.org/10.1007/s00253-014-6263-9