X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase fromMycobacterium tuberculosisduring the catalyzed reaction

Abstract

Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis (PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Å resolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt-ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on C(α) atoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme-substrate and enzyme-product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

Effect of macromolecular mass transport in microgravity protein crystallization

“…The first reported protein crystallization experiments in microgravity, performed in 1984, described the growth of lysozyme and b-galactosidase crystals on Spacelab-1 (Littke and John, 1984). Since then in subsequent space shuttle missions, unmanned satellite missions, on the Russian space station and on the International Space Station (ISS), more than 100 different proteins have been crystallized under microgravity conditions (DeLucas et al, 1986;Krauspenhaar et al, 2002;Snell et al, 1997;Timofeev et al, 2012a;Vallazza et al, 2002). In 1989, Asano et al performed a series of crystal growth experiments for bovine pancreatic ribonuclease S under microgravity conditions using the COSIMA 2 system (Asano et al, 1992;Plaas-Link and Cornier, 1988).…”

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

“…35−37 These studies, together with the availability of crystal structures of a number of CoA pathway enzymes for use in structure-guided drug design, provided further support for investigating the CoA pathway as an attractive source of new TB drug targets, encouraging the development of inhibitors against various pathway enzymes. 9,13,38−41 However, despite resource-intensive efforts that led to the identification of potent inhibitors of Mtb PanC and PanK enzymes, these molecules failed to translate into lead compounds with significant whole-cell activity. 13,42,43 Using cKD mutants of Mtb as tools to assess target vulnerability and the target selectivity of small-molecule inhibitors in whole Mtb cells, we 21 and others 43 concluded that these failures might be explained, at least in part, by the relative invulnerability of Mtb to depletion of PanC and PanK.…”

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase fromBurkholderia pseudomallei

“…In the MES‐bound form, the sulfate moiety binds in the oxyanion hole in the catalytic pocket. However, its binding is not enough to trigger the full reorientation of the C‐terminal helix as shown in the NTP binding forms ,…”