Kinase activity assays were performed in 96-well plates. Each activity measurement was performed in a final volume of 20 μl, containing 50 mM tris-HCl (pH 7.4), 0.1% (v/v) 2-mercaptoethanol, 0.01% (v/v) Brij-35, 2 mM MnCl2, 100 μM [γ-32P]ATP [5 to 50 counts per minute (cpm)/pmol], and 330 μM 17-mer peptide or 25 μM MtGarA as substrate. In addition, the kinase assays shown in Fig. 2F were done in the absence or presence of 92 μM MtGarAΔ43. The enzyme concentration in the assays was 0.2 to 5 μM and 3 to 12 nM when using the 17-mer peptide or MtGarA as substrates, respectively. The kinase reactions were started by the addition of 4 μl [γ-32P]ATP-Mn+2 and were performed at room temperature. The reactions were stopped by the addition of phosphoric acid, and 4 μl of each reaction was spotted on P81 phosphocellulose papers (Whatman) using the epMotion 5070 (Eppendorf) workstation. The papers were washed in 0.01% phosphoric acid, dried, and then measured and analyzed using a PhosphorImager (FLA-9000 Starion, Fujifilm). Each reaction was performed in duplicate (<10% variation), and each assay was performed at least twice (<20% variation). In all cases, specific activity values were derived from reactions performed using three different enzyme concentrations within the indicated ranges (<20% variation), verifying a linear dependence of activity with kinase concentration. The proportion of 17-mer peptide or MtGarA consumed in the reactions was lower than 10% and 30%, respectively. MtGarA consumption was verified to be linear in time up to 50% its initial concentration. Under the experimental conditions used to test phosphorylation of the 17-mer peptide or MtGarA, PknBCD or PknBCD,L33E autophosphorylation represented less than 5% of the total signal. The measured signal was at least five times higher than the lecture on the background.

Determinations of 2-oxoglutarate decarboxylase and ODH activities were carried out as previously described (21). Briefly, for the decarboxylase activity, reactions contained 2 μM MsKGDΔ360, 1 to 3 mM 2-oxoglutarate, 0.2 mM ThDP, 1 mM MgCl2, 0.55 to 1.1 μM MtGabD1, and 1 to 2 mM NADP+ (nicotinamide adenine dinucleotide phosphate) in 50 mM potassium phosphate (pH 6.5). Reactions were started by the addition of 2-oxoglutarate at 37°C, and initial rates were calculated during the linear phase of the reaction (10 to 20 min) following 340-nm absorbance using a microplate reader. For measuring ODH activity, the reaction contained 1 to 3 mM 2-oxoglutarate, 1 mM CoA-SH, 0.2 mM ThDP, 1 mM MgCl2, 2 μM MsKGD, 4 μM MsDlaT, 0.5 μM MsLpd, and 1 to 2 mM 3-acetylpyridine dinucleotide (AcNAD+) in 50 mM potassium phosphate (pH 6.5). AcNAD+ was used instead of NAD+ to prevent enzyme inhibition by NADH (reduced form of NAD+). Reactions were started by the addition of 2-oxoglutarate at 37°C, and initial rates were calculated during the linear phase of the reaction (10 to 20 min). When required, MsGarA was diluted in phosphate-buffered saline (PBS) and added to a final concentration ranging from 0.02 to 10 μM, and AcCoA to a final concentration ranging from 0.01 to 4 mM. The rate of AcNAD+ reduction was calculated by spectrophotometry based on the absorption coefficient of AcNADH,H+340nm = 6220 M−1 cm−1). When appropriate, MsGarA was diluted in PBS and added to the assay mixture at 5 μM, unless otherwise stated. Experiments were performed in triplicate.

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