AlaDH

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Alanine dehydrogenase (AlaDH) is currently used in various applications such as biotechnology, food and pharmaceuticals. The AlaDH (EC1.4.1.1) systematic name is L-alanine: NAD⁺ oxidoreductase (deamination), which belongs to oxidoreductase, because it reduces an electron acceptor NAD⁺/NADP⁺ in the process of oxidative deamination. AlaDH was first discovered by Wiame and Pierard in Bacillus subtilis. Subsequently, the kinetic properties and the catalytic limiting steps of the enzyme have been published in various microorganisms. Alanine dehydrogenase is different from glutamate dehydrogenase because it is not commonly produced in microorganisms, but occurs largely in Bacillus species.

The gene encoding AlaDH is called ald (alaD). The ald gene of M. tuberculosis has pyruvate aminating activity when expressed in E. coli. But when the ald gene is inactivated in M. tuberculosis, it will cause the loss of AlaDH function. In B. subtilis, aldR is a transcriptional activator, which can mediate ald expression and is important for sporulation. In Rhizobium leguminosarum, mutations in aldR prevent the induction of ald activity. Therefore, aldR is necessary for the induction of ald gene, and aldR can be used as either an activator or a repressor, depending on the presence or absence of L-alanine. Through DNase I footprint analysis, four aldR-binding sites (O2, O1, O4 and O3) were identified 95 bp upstream of the ald gene of M.smegmatis. O2, O1, and O4 are required for induction of ald expression by alanine, and O3 directly participates in inhibiting ald expression. Studies have shown that in the presence of alanine, the transcription regulator aldR changes its quaternary structure from a homodimer to an octamer with an open-ring conformation.

Figure 1. AlaDH-catalyzed forward and backward reaction (Dave, U.C; Kadeppagari, R.K. 2019)

Oxidative deamination activity

AlaDH catalyzes the removal of amino groups from L-alanine in a forward reaction, which can then decompose the carbon skeleton (pyruvate) by citric acid cycle. The role of AlaDH is to provide pyruvate as an energy source necessary for sporulation.

Reductive amination activity

Pyruvate and ammonium assimilated are metabolized by AlaDH to L-alanine. The main function of AlaDH in vivo is the reductive amination of pyruvate, which can be used to produce alanine to synthesize peptidoglycan. AlaDH plays an important role in the synthesis of the cell wall skeletons of microorganisms (such as Streptomyces aureofaciens) because L-alanine is one of the three essential amino acids that constitute repeating peptide subunits of the peptidoglycan. Because L-alanine is often present in many important known proteins, it also plays an important role in protein synthesis in the bacterial cytoplasm.

Figure 2. Function of AlaDH in microorganisms by oxidative deamination of L-alanine in forward reaction and reductive amination of pyruvate in backward reactions (Dave, U.C; Kadeppagari, R.K. 2019)

NAD⁺/NADH maintenance

AlaDH also plays an important role in the maintenance of the NAD⁺ pool. The ald knockout mutant of M. tuberculosis did not reduce the anaerobic survival in vitro, but it caused a significant delay in the resumption of growth following re-oxygenation. During the reactivation process, the NADH/NAD⁺ ratio of the mutant changed, which indicates that AlaDH is needed to maintain the optimal NADH/NAD⁺ ratio during anaerobiosis. AlaDH maintains the redox balance (NADH/NAD⁺) in M. tuberculosis during dormancy/hypoxia, and restores/reactivates when the oxygen level is increased enough to support the regeneration of M. tuberculosis.

Glyoxylate reductive aminase activity

Additionally, ald encodes a bifunctional AlaDH in some microorganisms. PvRA (pyruvate reductive aminase activity) catalyzes the reduction of pyruvate to alanine and GxRA (glyoxylate reductive aminase activity) catalyzes glyoxylate acid to glycine and oxidizes NADH to NAD+. AlaDH has been identified from many kinds of bacteria, but not all of these enzymes have GxRA activity, which may be due to the existence of the large sequence diversity in these enzymes. The dual function of AlaDH in Mycobacterium tuberculosis has been determined by biochemical and genetic methods. The inactivation of ald in M. tuberculosis leads to a decrease or loss of the activity of both PvRA and GxRA. The function was restored when the cloned copy of ald was then inserted into the genome. In addition, using pyruvate or glyoxylate as a substrate, a histidine-tagged protein purified from E. coli catalyzes these two reactions.

Figure 3. Reactions catalyzed by AlaDH (Dave, U.C; Kadeppagari, R.K. 2019)