Morpholine-induced formation of l -alanine dehydrogenase activity in Mycobacterium strain HE5

Schuffenhauer, Grit; Schräder, Thomas; Andreesen, Jan R.

doi:10.1007/s002030050728

Cited by 15 publications

(9 citation statements)

References 30 publications

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“…We also confirmed that expression of M. tuberculosis ald, as expressed in BCG, and the native chromosomal ald of M. marinum increases with decreasing ammonium concentration. Consistent with this, a recent study of a mycobacterial strain, HE5, showed that Lalanine dehydrogenase activity was not detectable in a bacterium grown on pyruvate and ammonium (5.6 mM), but was at its highest level when the bacterium was grown on L-alanine as the nitrogen source (32). These findings suggest that the primary role of L-alanine dehydrogenase is the oxidative deamination of L-alanine under nitrogen-limiting conditions.…”

Section: Discussionsupporting

confidence: 61%

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

et al. 2003

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Mycobacterium bovis BCG is the only accepted vaccine for the prevention of tuberculosis (TB) in humans.BCG is a live vaccine, and induction of immunity to TB requires productive infection of the host by BCG. However, BCG is not a satisfactory vaccine, because it fails to protect against pulmonary TB in adults. In this study, we found that BCG strains cannot utilize many naturally occurring amino acids as the sole nitrogen source for growth. This defect is caused, at least partially, by the lack of functional metabolic enzymes. All BCG strains are unable to catabolize L-alanine or D-alanine due to a frameshift mutation in the L-alanine dehydrogenase gene (ald). Some BCG strains, such as BCG-Pasteur and BCG-Frappier, cannot catabolize L-serine, apparently due to inadequate expression of L-serine deaminase (sdaA). We also found that undegraded alanine and serine inhibit the growth of BCG through blockage of glutamine synthetase. These results suggest that BCG strains are limited in nitrogen metabolic capacity and predict defects that may restrict multiplication and persistence of the live vaccine within the host.Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis isolated in 1921, is currently administered to over 100 million newborns per year as the only available vaccine against tuberculosis (TB) (13). BCG is only efficacious as a live vaccine; killed BCG does not provide protection in animal models, and killed Mycobacterium tuberculosis in human trials provided only weak and transient protection. In clinical trials of BCG vaccination, the observed efficacy has ranged from no protection to 80% fewer cases of TB (11, 13). A better understanding of this variable efficacy should help guide BCG programs and suggest avenues for the development of novel anti-TB vaccines.Several hypotheses have been generated to explain the vaccine trial data (3). The most prominent is that exposure to environmental mycobacteria partially sensitizes the host against mycobacteria and thereby provides heterologous immunity that obscures the potential benefits of BCG vaccination (14, 15). To support this, a recent study showed that the multiplication of the Danish strain of BCG was inhibited in animals previously sensitized with environmental mycobacteria. Consequently BCG vaccination elicited only a transient immune response and afforded no protection against a subsequent TB challenge (8). A second hypothesis involves differences in the vaccine strains used in clinical trials (9). After their introduction in 1921, BCG vaccines were maintained by in vitro passage in a variety of vaccine laboratories for 4 to 5 decades, resulting in phenotypic and genetic differences between BCG strains (6, 27). The capability of each of these BCG vaccines to protect against TB is unknown, because most clinical trials were performed with vaccine strains that have not been preserved. Notably, among genetic changes in BCG strains after 1921, one observes deletions in regulatory genes (which are postulated to govern the capacity to survi...

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Section: Discussionsupporting

confidence: 61%

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

et al. 2003

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“…Glutamate dehydrogenase provides a lower-affinity non-ATPrequiring system, but this activity was not detected in M. tuberculosis (50). Under certain conditions, alanine dehydrogenase may fulfill an ammonium assimilation role, as has been proposed for other bacteria (6,46). This could be important if glutamine synthetase is inhibited by high levels of alanine or glycine (9).…”

Section: Discussionmentioning

confidence: 91%

ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

Giffin

Modesti

Raab

et al. 2011

Journal of Bacteriology

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The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown. Mycobacterium tuberculosis is the causative agent of tuberculosis, and one of the most successful human pathogens. It was responsible for approximately 2 million deaths in 2008, while currently almost one-third of the world's population is infected with this organism. Research with M. tuberculosis has described a pathogen uniquely adapted to the wide range of harsh environments presented by the host. Much of this work has focused on the microbe's metabolism, with the idea of identification of novel enzymes or pathways to target for drug development.One of these environmental factors is nitrogen availability. Very little is known about the nitrogen sources used by M. tuberculosis in vivo. M. tuberculosis can utilize many amino acids for nitrogen, including alanine and glycine (29). Mutants of M. tuberculosis unable to synthesize proline retained the ability to replicate in the human macrophage cell line THP-1 (35), while other amino acid auxotroph mutants were attenuated (3,17,22,52). A Mycobacterium bovis BCG mutant unable to make methionine showed survival in mice similar to the wild-type strain (32). This suggests some amino acids are available in vivo and serve as nutrients for M. tuberculosis.The enzyme glycine dehydrogenase was first described in M. tuberculosis in 1962 (16). This enzyme was detected by the reductive amination of glyoxylate to glycine concurrent with the oxidation of NADH to NAD ϩ (Fig. 1). This reaction represents glyoxylate reductive aminase (GxRA) activity. The activity corresponding to the reverse reaction, catalyzed by glycine dehydrogenase (GDH), was not detected. The expression of glyoxylate reductive amination by a putative glycine dehydrogenase in M. tuberculosis has been characterized in nonreplicating persistent (NRP) cultures (58). In the Wayne model of dormancy, sealed cultures of M. tuberculosis create a microaerobic environment (NRP-1), which subsequently develops into the anaerobic stage (NRP-2) (58). GxRA activity w...

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“…The alanine dehydrogenase from Mycobacterium strain HE5 seems to be regulated by different factors. A high enzyme activity was only detected in cells grown with nitrogencontaining substrates, but not with pyruvate plus ammonia, and the oxidative deamination of L-alanine was significantly stimulated by morpholine (Schuffenhauer et al 1999).…”

Section: Discussionmentioning

confidence: 94%

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

Laue

Cook

2000

Archives of Microbiology

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Alanine dehydrogenase [L-alanine:NAD + oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent K m values for alanine, NAD + , pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.

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Morpholine-induced formation of l -alanine dehydrogenase activity in Mycobacterium strain HE5

Cited by 15 publications

References 30 publications

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

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