Meneely 2015

Structure informs on function: A thiazolinyl imine reductase of siderophore biosynthesis

Kathleen M. Meneely1, Andrew P. Riley2, Thomas E. Prisinzano2,3, Audrey L. Lamb1

 Molecular Biosciences1, Chemistry2 and Medicinal Chemistry3, University of Kansas, Lawrence, KS

Iron is an essential element for most pathogenic bacteria that must obtain this nutrient from the environment. Many of these bacteria acquire iron by synthesizing low molecular weight chelating molecules termed siderophores. Yersiniabactin is produced by Yersinia enterocolitica in iron-limiting environments and is composed of salicylate, three cyclized cysteines and one malonyl moiety. The thiazolinyl imine reductase (Irp3) catalyzes the NADPH-dependent reduction of one of the three thiazoline rings of a yersiniabactin intermediate. The apo form of Irp3 was solved to 1.85 Å resolution by selenomethionine MAD with 23 seleniums in the asymmetric unit and a final Rcryst of 19.6% and an Rfree of 24.2%. Irp3 is a homodimer consisting of two domains, an N-terminal NADPH binding domain and a C-terminal dimerization domain and shows structural similarity to sugar oxidoreductases and biliverdin reductase. An NADP+ bound form of Irp3 was determined by molecular replacement to 2.31 Å resolution with apo-Irp3 as the model and final Rcryst and Rfree of 19.7% and 26.0%, respectively. The NADP+-bound Irp3 structure shows clear density for NADP+ in each of the four monomers of the asymmetric unit. Four more recent holo structures have been determined with NADP+, NADP+ and a substrate analog, NADPH and a substrate analog, and NADP+ with a product analog to 1.28 – 1.7 Å resolution. A reaction mechanism is proposed for the donation of a proton from a general base (either His101 or Tyr128) and hydride donation from the C4 of the NADPH cofactor for the reduction of the carbon-nitrogen double bond of the thiazoline ring. A homology model of the functionally similar thiazolinyl reductase in Pseudomonas aeruginosa (PchG) was generated. An extensive loop is found in Irp3 and PchG but not in other structural homologues. This loop is hypothesized to be involved in binding to the nonribosomal peptide synthetase modules to which the substrate is covalently attached. A kinetic analysis monitoring NADPH oxidation using a substrate analog and an HPLC assay is presented.