Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth

Zhengyu Feng, Nancy E. Cáceres, Gautam Sarath, Raul G Barletta

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Aid) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Aid 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 Aid 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 Aid plays an important role in both alanine utilization and anaerobic growth.

Original languageEnglish (US)
Pages (from-to)5001-5010
Number of pages10
JournalJournal of bacteriology
Volume184
Issue number18
DOIs
StatePublished - Sep 1 2002

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Alanine Dehydrogenase
Mycobacterium smegmatis
Alanine
Nitrogen
Glycine Dehydrogenase
Growth
Amination
Enzymes
Oxygen
Deamination
Pyruvic Acid
NAD
Proteins
Phenotype
Food

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth. / Feng, Zhengyu; Cáceres, Nancy E.; Sarath, Gautam; Barletta, Raul G.

In: Journal of bacteriology, Vol. 184, No. 18, 01.09.2002, p. 5001-5010.

Research output: Contribution to journalArticle

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abstract = "NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Aid) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Aid 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 Aid 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 Aid plays an important role in both alanine utilization and anaerobic growth.",
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