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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.Graphical abstract

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.

Copalyl diphosphate synthase from (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 Å-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity.

Lysyl oxidase-2 (LOXL2) is a Cu 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 Zn -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 Cu 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 Cu . We propose that LTQ resides within 2.9 Å of the active site of Cu in the mature LOXL2, and both LTQ and Cu are solvent-exposed.

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.