Abstract
A link between carbon and nitrogen metabolism is important for serving as metabolic ancillary reactions. Here, we identified and characterized the alanine dehydrogenase gene in Aphanothece halophytica (ApalaDH) that is involved in alanine assimilation/dissimilation. Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to l-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of l-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity. Kinetic analysis showed the lowest affinity for pyruvate followed by l-alanine and glyoxylate with a Km of 0.22 ± 0.02, 0.72 ± 0.04, and 1.91 ± 0.43 mM, respectively. ApalaDH expression was upregulated by salt. Only PvRA and GxRA activities were detected in vivo and both activities increased about 1.2- and 2.7-fold upon salt stress. These features implicate that the assimilatory/dissimilatory roles of ApAlaDH are not only selective for l-alanine and pyruvate, but also, upon salt stress, can catabolize glyoxylate to generate glycine.
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Abbreviations
- ADH:
-
Alanine dehydrogenase
- PvRA:
-
Pyruvate reductive aminase activity
- ALD:
-
Alanine oxidative dehydrogenase activity
- GxRA:
-
Glyoxylate reductive aminase activity
- GDH:
-
Glycine oxidative dehydrogenase activity
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Acknowledgements
This study was funded by the research fund of the Research Institute of Meijo University and the Chemical Approaches for Food Applications Research Group, Faculty of Science, Chulalongkorn University.
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Communicated by Erko Stackebrandt.
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Figure 1S. Effects of NaCl and KCl on ApAlaDH activity
. The following ApAlaDH enzymatic activities were assayed in the presence or absence of NaCl or KCl: A) and D) PvRA, B) and E) ALD, and C) and F) GxRA. The ApAlaDH activity measured in the absence of NaCl or KCl was set to 100%. The data is represented as the mean ± standard deviation from three independent experiments. (PPT 215 KB)
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Phogosee, S., Hibino, T., Kageyama, H. et al. Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties. Arch Microbiol 200, 719–727 (2018). https://doi.org/10.1007/s00203-018-1481-7
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DOI: https://doi.org/10.1007/s00203-018-1481-7