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. 2012 Mar;194(5):1045-54.
doi: 10.1128/JB.05914-11. Epub 2011 Dec 30.

ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase

Michelle M Giffin  1 , Lucia Modesti, Ronald W Raab, Lawrence G Wayne, Charles D Sohaskey
Affiliations

ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase

Michelle M Giffin et al. J Bacteriol. 2012 Mar.

Abstract

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.

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Figures

Fig 1
Fig 1
Possible reactions of alanine dehydrogenase. GDH activity was not detected.
Fig 2
Fig 2
Southern blot analyses of the ald insertional mutant and complement. (A) DNA was isolated from each strain, cut with SacI, and analyzed with a probe specific for ald. RVW7, Δald::hyg; Ald21, RVW7(pAld-Gen21). The approximate sizes are indicated on the left. (B) Gene organization of ald, showing the insertion of hyg. Arrows indicate open readings frames and the direction of transcription. X, XbaI site; B, BamHI site; S, SacI site. ald′ is the disrupted ald. The ald probe binding site is shown.
Fig 3
Fig 3
(A and B) NADH breakdown with glyoxylate in aerobic cultures (A) or nonreplicating persistent stage 2 cultures (B). Filled symbols and solid lines show data for complete reactions, while empty symbols and dotted lines are for reactions without ammonium sulfate. Circles, WT; triangles, RVW7; squares, RVW7(pAld-Gen21). The standard deviations are shown. (C and D) PvRA and GxRA activities of the Δald mutant in aerobic cultures (C) or nonreplicating persistent stage 2 cultures (D). PvRA is involved in the reductive amination of pyruvate to alanine. GxRA is involved in the reductive amination of glyoxylate to glycine. RVW7, Δald; Ald21, RVW7(pAld-Gen21). Specific activity is reported in μmoles of NADH oxidized/min/μg of protein. The standard deviations are shown.
Fig 4
Fig 4
Activity of His-tagged Ald at different pH levels. (A) Pyruvate reductive amination activity (circles) and alanine dehydrogenase activity (squares). (B) Glyoxylate reductive amination activity (circles), glycine dehydrogenase activity (squares), and glyoxylate reductive amination activity with glutamine in place of ammonium sulfate (triangles). Specific activity is shown in μmoles of NADH oxidized/min/μg of protein. The standard deviations are shown.
Fig 5
Fig 5
Activity of the Ald enzyme with alternate substrates. (A) Reductive amination reaction with pyruvate (Pyv), glyoxylate (Glx), α-ketoglutarate (aKT), hydroxypyruvate (Hpv), malate (Mal), acetate (Ace), glycolate (Glc), oxaloacetate (Oaa), and methylglyoxal (Mg). (B) Oxidative deamination reaction with alanine (Ala), glutamate (Glu), proline (Pro), aspartate (Asp), serine (Ser), and glycine (Gly). The specific activity of the His-tagged Ald is reported in μmoles of NADH oxidized/min/μg of protein. The standard deviations are shown.
Fig 6
Fig 6
Subcellular location of Ald. Cell extracts and culture filtrates from aerobically growing or NRP-2 cultures were probed with an anti-Ald antibody. (A) AG, aerobically growing cells; NRP, nonreplicating persistent stage 2 cells; F, growth medium filtrate; E, cell extracts. (B) W, cell wall fraction; S, soluble fraction; M, cell membrane.
Fig 7
Fig 7
Growth of RVW7. (A) Growth of M. tuberculosis in LIM with different nitrogen sources. Circles, asparagine; squares, NH4Cl; diamonds, glycine. WT samples, solid lines with filled symbols; RVW7, dotted lines with empty symbols. (B) Growth in LIM with alanine. Circles, WT; squares, RVW7; triangles, RVW7(pAld-Gen21). The standard deviations are shown.
Fig 8
Fig 8
Quantitative real-time PCR of ald grown with different nitrogen sources. RNA levels are expressed relative to stable 16S rRNA. (Insert) Expression of Rv0239. The standard deviations are shown.
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