Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica : characterization and m
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ORIGINAL PAPER
Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties Sittipol Phogosee1 · Takashi Hibino2 · Hakuto Kageyama2 · Rungaroon Waditee‑Sirisattha1 Received: 13 November 2017 / Revised: 11 January 2018 / Accepted: 18 January 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018
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. Keywords Cyanobacteria · Alanine dehydrogenase · Aphanothece halophytica Abbreviations ADH Alanine dehydrogenase PvRA Pyruvate reductive aminase activity ALD Alanine oxidative dehydrogenase activity GxRA Glyoxylate reductive aminase activity GDH Glycine oxidative dehydrogenase activity
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00203-018-1481-7) contains supplementary material, which is available to authorized users. * Hakuto Kageyama kageyama@meijo‑u.ac.jp * Rungaroon Waditee‑Sirisattha [email protected] 1
Department of Microbiology, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
Graduate School of Environmental and Human Sciences, Meijo University, Nagoya 468‑8502, Japan
2
Introduction Alanine dehydrogenase (AlaDH) catalyzes the NADdependent reversible reaction of l -alanine to pyruvate (PvRA ↔ ALD) (Fig. 1a). This enzyme has an important role in both assimilatory and dissimilatory metabolic functions. To date, several lines of evidence have shown that AlaDHs are involved in various physiological processes, such as growth, sporulation and heterocyst formation. Among living organisms, bacterial AlaDHs have been extensively studied. Malfunction of AlaDH resulted in bacterial pathophysiology, with impacts on sporulation and heterocyst formation (Giffin et al. 2012; Siranosian et al. 1993; Pernil et al. 2010). Specif
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