PMID:12193615

Citation	Feng, Z, Cáceres, NE, Sarath, G and Barletta, RG (2002) Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth. J. Bacteriol. 184:5001-10
Abstract	NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.
Links	PubMed PMC135311
Keywords	Alanine/metabolism; Alanine Dehydrogenase; Amino Acid Oxidoreductases/genetics; Amino Acid Oxidoreductases/metabolism; Anaerobiosis; Base Sequence; Cloning, Molecular; Culture Media; Molecular Sequence Data; Mutation; Mycobacterium smegmatis/enzymology; Mycobacterium smegmatis/growth & development; Nitrogen/metabolism; Oxygen/pharmacology; Sequence Analysis, DNA
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MYCSM:DHA	enables	GO:0000286: alanine dehydrogenase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
MYCSM:DHA	enables	GO:0000286: alanine dehydrogenase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
MYCSM:DHA		GO:0000286: alanine dehydrogenase activity	ECO:0000315:		F		Figure 6b: Shows the utilization of nitrogen sources by M. smegmatis wild-type, ald null mutant, and complemented strains. Exponentially of each strain were harvested, washed, and inoculated into minimal medium containing L-alanine as the sole nitrogen source. More viable cells were produced in the wild type strain compared to mutant and complemented strains. This is because alanine dehydrogenase catalyzes a reaction that generates nitrogen from L-alanine.	complete CACAO 9692

References

See Help:References for how to manage references in GONUTS.