An Openand Shut Case: The Interaction of Magnesiumwith MST Enzymes

摘要

The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthaseenzymes but not the salicylate synthase enzymes. Prior structuraldata have suggested that binding of the metal ion occludes accessor egress of substrates. Our kinetic data indicate that for the productionof isochorismate, a high magnesium ion concentration suppresses therate of release of product, accounting for the observed inhibitionand establishing the basis of the ordered-addition kinetic mechanism.Moreover, we show that isochorismate is channeled through the synthasereaction as an intermediate that is retained in the active site bythe magnesium ion. Indeed, the lyase-active enzyme has 3 orders ofmagnitude higher affinity for the isochorismate complex relative tothe chorismate complex. Apparent negative-feedback inhibition by ferrousions is documented at nanomolar concentrations, which is a potentiallyphysiologically relevant mode of regulation for siderophore biosynthesisin vivo.