Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Abstract

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Precipitant–ligand exchange technique reveals the ADP binding mode inMycobacterium tuberculosisdethiobiotin synthetase

“…4b and 5b) the DAPA binding site. In both conformations the ADP -phosphate bound at the same position as that observed for CTP (Salaemae et al, 2015), in a highly coordinated network of hydrogen bonds and electrostatic interactions with the P-loop, involving the backbone amides of Val13 (3.2-3.42 Å ), Gly14 (2.67-3.15 Å ), Lys15 (2.47-3.05 Å ) and Thr16 (3.04-3.1 Å ), as well as the side chains of Lys15 (2.55-2.65 Å ) and Thr16 (2.91-3.07 Å ) ( Figs. 5a and 5b).…”

“…MtDTBS was expressed and purified as described previously (Salaemae et al, 2015), except that a HiPrep 26/60 Sephacryl S-300 HR column (GE Healthcare) was used as an additional purification step following IMAC chromatography. MtDTBS was concentrated to 10 mg ml À1 and stored in the following solution: 25 mM Tris pH 7.5, 30 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 5%(v/v) glycerol.…”

“…Thus, ADP provides a potential starting point for the chemical development of nucleoside analogues to treat antibiotic-resistant tuberculosis. The crystal structure of MtDTBS in complex with cytidine triphosphate (CTP; PDB entry 4wop) revealed the binding mode for this ligand (Salaemae et al, 2015). The cytosine moiety bound in a pocket formed by the coils connecting 7 to 3 10 2 and 6 to 7 (Figs.…”

“…However, efforts to crystallize MtDTBS in complex with other nucleosides such as ADP have been unsuccessful ISSN 2059-7983 # 2018 International Union of Crystallography (Salaemae et al, 2015;Dey et al, 2010) despite similar structures having been obtained with homologues from other bacteria (Huang et al, 1995;Kä ck et al, 1998). In our repeated attempts to solve the ADP-MtDTBS crystal structure, electron density consistent with a sulfate ion bound at the P-loop was observed, resulting from the high concentrations of ammonium sulfate in the crystallization condition (1.2-1.7 M ammonium sulfate, 0.1 M Tris pH 8, 10-15% glycerol; adapted from Salaemae et al, 2015). Concordant with this, a sulfate ion was observed instead of CTP in one active site of the CTP-MtDTBS crystal structure (Salaemae et al, 2015).…”

“…In our repeated attempts to solve the ADP-MtDTBS crystal structure, electron density consistent with a sulfate ion bound at the P-loop was observed, resulting from the high concentrations of ammonium sulfate in the crystallization condition (1.2-1.7 M ammonium sulfate, 0.1 M Tris pH 8, 10-15% glycerol; adapted from Salaemae et al, 2015). Concordant with this, a sulfate ion was observed instead of CTP in one active site of the CTP-MtDTBS crystal structure (Salaemae et al, 2015). We propose that precipitants that are present at high concentrations in the crystallization condition directly compete with ADP for binding to the P-loop.…”