Allosteric pyruvate kinase-based "logic gate" synergistically senses energy and sugar levels in <i>Mycobacterium tuberculosis</i>

Pyruvate kinase (PYK) is an essential glycolytic enzyme that controls glycolytic flux and is critical for ATP production in all organisms, with tight regulation by multiple metabolites. Yet the allosteric mechanisms governing PYK activity in bacterial pathogens are poorly understood. Here we report biochemical, structural and metabolomic evidence that Mycobacterium tuberculosis (Mtb) PYK uses AMP and glucose-6-phosphate (G6P) as synergistic allosteric activators that function as a molecular "OR logic gate" to tightly regulate energy and glucose metabolism. G6P was found to bind to a previously unknown site adjacent to the canonical site for AMP. Kinetic data and structural network analysis further show that AMP and G6P work synergistically as allosteric activators. Importantly, metabolome profiling in the Mtb surrogate, Mycobacterium bovis BCG, reveals significant changes in AMP and G6P levels during nutrient deprivation, which provides insights into how a PYK OR gate would function during the stress of Mtb infection

The catalytic pathway of horseradish peroxidase at high resolution

A molecular description of oxygen and peroxide activation in biological systems is difficult, because electrons liberated during X-ray data collection reduce the active centres of redox enzymes catalysing these reactions(1-5). Here we describe an effective strategy to obtain crystal structures for high-valency redox intermediates and present a three-dimensional movie of the X-ray-driven catalytic reduction of a bound dioxygen species in horseradish peroxidase (HRP). We also describe separate experiments in which high-resolution structures could be obtained for all five oxidation states of HRP, showing such structures with preserved redox states for the first time

Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family

Effectors secreted by the type Ill secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6