Heterologous Expression and Partial Characterization of a New Alanine Dehydrogenase from Amycolatopsis sulphurea.

A novel alanine dehydrogenase (AlaDH; EC.1.4.1.1) was isolated from Amycolatopsis sulphurea and the AlaDH gene was cloned into a pET28a(+) plasmid and expressed in E. coli BL21 (DE3). The molecular mass of this enzyme was calculated as 41.09 kDa and the amino acid residues of the pure protein indicated the presence of N terminus polyhistidine tags. Its enzyme kinetic values were Km 2.03 mM, kcat 13.24 (s-1), and kcat/Km 6.53 (s-1 mM-1). AlaDH catalyzes the reversible conversion of L-alanine and pyruvate, which has an important role in the TCA energy cycle. Maximum AlaDH activity occurred at about pH 10.5 and 25 °C for the oxidative deamination of L-alanine. AlaDH retained about 10% of its relative activity at 55 °C and it remained about 90% active at 50 °C. These findings show that the AsAlaDH from A. sulphurea has the ability to produce valuable molecules for various industrial purposes and could represent a new potential biocatalyst for biotechnological applications after further characterization and improvement of its catalytic properties.

Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties.

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.

AdeR, a PucR-type transcription factor, activates expression of L-alanine dehydrogenase and is required for sporulation of Bacillus subtilis.

The Bacillus subtilis ald gene encodes L-alanine dehydrogenase, which catalyzes the NAD(+)-dependent deamination of L-alanine to pyruvate for the generation of energy and is required for normal sporulation. The transcription of ald is induced by alanine, but the mechanism underlying alanine induction remains unknown. Here we report that a gene (formerly yukF and now designated adeR) located upstream of ald is essential for the basal and alanine-inducible expression of ald. The disruption of the adeR gene caused a sporulation defect, whereas the complementation of an adeR mutation with an intact adeR gene restored the sporulation ability. adeR expression was not subject to autoregulation and alanine induction. Deletion and mutation analyses revealed that an inverted repeat, centered at position -74.5 relative to the transcriptional initiation site of ald, was required for ald expression and also likely served as a ρ-independent transcription terminator. Electrophoretic mobility shift assays showed that purified His-tagged AdeR was a specific DNA-binding protein and that this inverted repeat was required for AdeR binding. AdeR shows no significant amino acid sequence similarity to the known transcriptional activators of ald genes from other bacteria. AdeR appears to be a member of the PucR family of transcriptional regulators. Its orthologs of unknown function are present in some other Bacillus species. Collectively, these findings support the notion that AdeR is a transcriptional activator which mediates ald expression in response to alanine availability and is important for normal sporulation in B. subtilis.