ADENYLATION AND TAILORING ACTIVITIES IN THE NONRIBOSOMAL PEPTIDE SYNTHESIS OF THE SIDEROPHORE PYOCHELIN

Pathogenic bacteria are becoming increasingly resistant to antibiotics. In response to this alarming trend, the scientific community must determine therapeutic targets. Essential nutrients, such as iron, are necessary for pathogens to survive and become virulent within a host system. One mechanism used by pathogenic bacteria to acquire iron from its surrounding environment is to produce low-molecular weight compounds which have a high affinity towards ferric iron. These compounds are called siderophores, and studies have shown their production to be essential for growth and virulence of some pathogens in iron-limited environments, such as the human host. Siderophores are often biosynthesized by nonribosomal peptide synthetases (NRPSs), which rarely have human homologs, making them attractive targets for novel therapeutics. NRPSs are enzymes utilized by bacteria, fungi, and plants to generate bioactive peptides. These bioactive peptides are not only used as secondary metabolites (toxins, pigments, siderophores) but have also found their way into the clinic as antibiotics, anticancer drugs, and immunosuppressants. To elicit their unique bioactivity, these peptides are tailored, making the compound chemically unique. Natural product chemists, metabolic engineers, and researchers in biochemistry and biotechnology work to exploit NRPS biosynthesis to generate new compounds for clinical use. This dissertation describes mechanistic and structural analyses of the adenylation and tailoring domains of the NRPS biosystem responsible for the production of pyochelin, a siderophore produced by antibiotic resistant Pseudomonas aeruginosa. A large portion of this work aims to better understand adenylation and “stuffed” tailoring didomains which lack structural characterization and have limited mechanistic understanding yet are ubiquitous in NRPS bioassembly. Pyochelin biosynthesis employs an adenylation-epimerase stuffed didomain in PchE and an adenylation-methyltransferase stuffed didomain in PchF. Substrate and product analogs were synthesized and steady-state adenylation, epimerase, and methyltransferase assays, along with onium chalcogen effects of the methyltransferase reaction, were used to characterize the adenylation-tailoring stuffed domains in pyochelin bioassembly. Similarly, substrate analogs were generated and used in steady-state kinetic and crystallography experiments with the stand-alone tailoring, NADPH-dependent reductase, PchG, and homolog, Irp3, of yersiniabactin biosynthesis. Finally, a steady-state adenylation assay was developed for the stand-alone salicylate adenylation enzyme, PchD, and potential warhead inhibitors were synthesized and co-crystallized laying the groundwork for future inhibitor design

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5′-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed

Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis

Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (<i>Yersinia</i> spp.) and pyochelin (<i>Pseudomonas aeruginosa</i>). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases. Two closed structures of the thiazolinyl imine reductase from <i>Yersinia enterocolitica</i> (Irp3) are presented here: an NADP⁺-bound structure to 1.45 Å resolution and a holo structure to 1.28 Å resolution with NADP⁺ and a substrate analogue bound. Michaelis–Menten kinetics were measured using the same substrate analogue and the homologue from <i>P. aeruginosa</i>, PchG. The data presented here support the hypothesis that tyrosine 128 is the likely general acid residue for catalysis and also highlight the phosphopantetheine tunnel for tethering of the substrate to the nonribosomal peptide synthetase module during assembly line biosynthesis of the siderophore