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A periplasmic, pyridoxal-5′-phosphate-dependent amino acid racemase in Pseudomonas taetrolens

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Abstract

The pyridoxal-5′-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.

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Acknowledgement

We thank A. Tauch (Bielefeld University, Germany) for support with preparation of integration mutants, Y. Gougami (Kansai University) for support with sequencing, and T. Polen (Forschungszentrum Juelich) for help with automatic blast analyses.

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu, 564-8680, Japan

    Daisuke Matsui, Tadao Oikawa, Noriaki Arakawa & Shintaro Osumi

  2. Institute of Biotechnology 1, Forschungszentrum Juelich, 52425, Juelich, Germany

    Frank Lausberg, Norma Stäbler, Roland Freudl & Lothar Eggeling

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  1. Daisuke Matsui
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  2. Tadao Oikawa
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  3. Noriaki Arakawa
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  4. Shintaro Osumi
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  5. Frank Lausberg
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  6. Norma Stäbler
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  7. Roland Freudl
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  8. Lothar Eggeling
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Correspondence to Tadao Oikawa.

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Matsui, D., Oikawa, T., Arakawa, N. et al. A periplasmic, pyridoxal-5′-phosphate-dependent amino acid racemase in Pseudomonas taetrolens . Appl Microbiol Biotechnol 83, 1045–1054 (2009). https://doi.org/10.1007/s00253-009-1942-7

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  • DOI: https://doi.org/10.1007/s00253-009-1942-7

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