Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo- (1.85 Å) and NADP+-bound (2.31 Å) forms. Irp3 shows structural homology to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-D-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologs that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism of proton donation from a general acid (either histidine-101 or tyrosine-128) and hydride donation from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline

Crystal Structure of Firefly Luciferase in a Second Catalytic Conformation Supports a Domain Alternation Mechanism

Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of the luciferin substrate, followed by an oxidative decarboxylation that ultimately produces light. Evidence for homologous acyl-CoA synthetases supports a domain alternation catalytic mechanism in which these enzymes’ C-terminal domain rotates by ∼140° to adopt two conformations that are used to catalyze the two partial reactions. While many structures exist of acyl-CoA synthetases in both conformations, to date only biochemical evidence supports domain alternation with luciferase. We have determined the structure of a cross-linked luciferase enzyme that is trapped in the second conformation. This new structure supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of the luciferase oxidative step

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

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 (<i>m</i>enaquinone, <i>s</i>iderophore, and <i>t</i>ryptophan 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 synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo