Structural and molecular dynamics ofMycobacterium tuberculosismalic enzyme, a potential anti-TB drug target

Abstract

<jats:title>Abstract</jats:title><jats:p>Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, <jats:italic>Mycobacterium tuberculosis</jats:italic> (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ provides the required reducing power for lipid biosynthesis. Here, we present the X-ray crystal structure of MEZ to 3.6 Å resolution and compare it with known structures of prokaryotic and eukaryotic malic enzymes. We use biochemical assays to determine its oligomeric state and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn<jats:sup>2+</jats:sup> and NAD(P)<jats:sup>+</jats:sup>, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually disordered, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that MEZ subunits alternate between open and closed states and that MEZ can stably bind its NAD(P)<jats:sup>+</jats:sup> cofactor in multiple conformations, including an inactive, compact NAD<jats:sup>+</jats:sup> form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb.</jats:p>

Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits from the GSK Chagas Box

“…Inspection of the solvent accessible surface showed that the ligand's binding site is completely buried into the dimer interface, without evident access to the solvent and distant from the substrate or cofactor binding sites, as described for other MEs [12][13][14][15][16]28,29,31 (Figure 2C). Consequently, local structural adjustments would be required for the ligand to access its binding pocket.…”

“…Contrary to the majority of the eukaryotic large unit MEs, TcMEc does not have N-nor C-terminal extensions that are known to stabilize the tetrameric form of the enzyme 14,16,28,31 (Figure S1). Further, TcMEc showed an estimated molecular mass equivalent to a dimer in size exclusion chromatography (SEC) experiments (Figure S2), which is in agreement with previous studies.…”

Mycobacterium tuberculosis carbon and nitrogen metabolic fluxes

“…Mtb lacking MEZ displayed altered cell wall composition and attenuated entry into macrophages (39,40). Mtb lacking PPDK had significantly reduced survival upon BDQ treatment compared to the wild type posing PPDK as an attractive drug target (36).…”