Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei

Abstract

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4'-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B. pseudomallei with the expectation that, because of the importance of the enzyme in coenzyme A biosynthesis, they will aid in the search for defenses against this pathogen. A crystal grown in ammonium sulfate yielded a 2.1 Å resolution structure that contained dephospho-coenzyme A with partial occupancy. The overall structure and ligand-binding interactions are quite similar to other bacterial PPAT crystal structures. A crystal grown at low pH in the presence of coenzyme A yielded a 1.6 Å resolution structure in the same crystal form. However, the experimental electron density was not reflective of fully ordered coenzyme A, but rather was only reflective of an ordered 4'-diphosphopantetheine moiety.

Figure 1

Multiple sequence alignment of bacterial PPATs. Sequences are shown from B. pseudomallei (Bp PPAT; PDB entry 3pxu; present study), E. coli (PDB entry 1b6t; Izard & Geerlof, 1999 ▶), Y. pestis (PDB entry 3l93; Osipiuk et al., unpublished work), T. maritima (PDB entry 1vlh; Joint Center for Structural Genomics, unpublished work), M. tuberculosis (PDB entry 1tfu; Morris & Izard, 2004 ▶), B. subtilis (PDB entry 1o6b; Badger et al., 2005 ▶) and T. thermophilus (PDB entry 1od6; Takahashi et al., 2004 ▶). α-­Helices and β-sheets from the Bp PPAT structure are shown as magenta cylinders and yellow arrows, respectively. The side chains of Thr9, Arg87 and Glu98 interact with dephospho-coenzyme A in the 3pxu structure. This figure was prepared with Geneious (Drummond et al., 2010 ▶).

Figure 2

Crystal structure of Bp PPAT solved at 2.1 Å resolution. (a) Biologically relevant hexameric structure of Bp PPAT from a crystal structure with bound dephospho-coenzyme A solved at 2.1 Å resolution. (b) Overlay of the dephospho-coenzyme A-bound structures of Bp PPAT (green) and E. coli PPAT (PDB entry 1b6t, magenta; Izard & Geerlof, 1999 ▶). Green spheres are used to illustrate disordered residues in the Bp PPAT crystal structures. Figs. 2 and 3 were prepared using PyMOL (DeLano, 2002 ▶).

Figure 3

Crystal structure of Bp PPAT solved at 1.6 Å resolution. (a) Monomeric structure of Bp PPAT in ribbon format with molecular-surface rendering in light transparency. Active-site components are modelled in stick representation and selected waters are shown as spheres. The active-site ligand-omit density map (|F _o| − |F _c|) is shown as a green mesh contoured at 3.0σ. (b) Close-up of the active site. The final protein model is shown in ribbon representation along with 4′-diphosphopantetheine, adenine and a sulfate ion in stick representation and selected waters as red spheres. The active-site ligand-omit electron-density map (|F _o| − |F _c|) is shown as a green mesh contoured at 3.0σ. (c) Active site as in (b) with the 2|F _o| − |F _c| electron-density map shown as a blue mesh contoured at 1.0σ. (d) Overlay of the final refined structure of Bp PPAT from (c) along with the structure of Bp PPAT-bound dephospho-coenzyme A solved at 2.1 Å resolution with coloring as in Fig. 2 ▶(b).