Natural products (NPs) isolated from fungi in the Trichoderma genus have significant agricultural applications as fertilizers and herbicides.[1] One class of these bioactive NPs are aromatic polyketides synthesized by multidomain non-reducing polyketide synthase (NRPKS) enzymes.[2] Within a typical fungal NRPKS, regioselective first-ring cyclization of the polyketide chain is controlled by the product template (PT) domain.[3] One limitation of current NP discovery methods is the dormancy of certain biosynthetic gene clusters (BGCs) under standard laboratory conditions, which can be activated through the variation of growth conditions.[1] This method was used to activate a four-gene BGC of Trichoderma afroharzianum T22 with an unusual NRPKS (MecA) as the core enzyme. The role of MecA in the biosynthesis of the final chromone compound produced by this BGC is explored in this project. The MecA-PT active site contains mutated catalytic residues, suggesting that first-ring cyclization occurs independently of the PT domain. Mutagenesis and in vitro domain dissection studies of MecA confirm that the PT domain is not involved in this pathway, with further studies needed to determine the exact cyclization mechanism. New layers of polyketide chemistry were revealed through this project, creating further appreciation for the diversity of natural product biosynthesis. References: [1] Han, W., et al. (2023). J. Agric. Food Chem. [2] Zhou, H., et al. (2010). Natural Product Reports. [3] Crawford, J. M., et al. (2009). Nature.

The development of stable, nonaqueous electrolytes remains a critical challenge in electrochemistry, as most available tetraalkylammonium salts contain beta-hydrogens that lead to decomposition under basic conditions, limiting their utility in stabilizing reactive anions. Here, we report a series of novel beta-hydrogen elimination-resistant tetraalkylammonium electrolytes that are soluble in common nonaqueous solvents and exhibit enhanced basic stability. Using the TEMPO/TEMPO⁻ redox couple as a mechanistic probe, our electrolytes enable previously irreversible redox events to proceed with substantially enhanced reversibility under standard conditions, while still maintaining high stability and solubility in common nonaqueous solvents. Conductivity measurements confirm that the salts maintain practical ion mobility in solution. These results address the critical gap in nonaqueous electrolyte chemistry by providing robust, soluble, and base-resistant supporting electrolytes suitable for mechanistic studies and potential applications in electrosynthesis and energy storage systems.

The application of electron diffraction to microcrystals (microED) complements routine use of X-ray diffraction (XRD) for the structural characterization of racemic and mixed-chirality peptide systems. These include peptidic assemblies that form rippled β-sheets, a novel secondary structure composed of alternating L- and D-amino acid β-strands. I will discuss the atomic-resolution characterization of two of these systems: the racemic, linear peptide NFGGILS/nfggils and the mixed-chirality, cyclic peptide FKFGGfdfgg, whose uppercase and lowercase letters denote L-and D- amino acids respectively, while “/” indicates a 1:1 racemic mixture of linear enantiomeric peptides. My analyses indicate that both structures possess canonical rippled β-sheet features. Notably, the NFGGILS/nfggils system displays a high-degree of planarity across its glycine residues, while FKFGGfdfgg exhibits an atypical distortion of its cyclic backbone driven in part by side-chain-to-main-chain hydrogen bonding. Subsequent and ongoing efforts have targeted additional systems: including amyloid-derived sequences and synthetic cyclic architectures. Collectively, these studies expand the repertoire of rippled β-sheet-forming peptides and provide insight into sequence- and environment-dependent determinants of ripple formation.

The metalloenzyme carbon monoxide dehydrogenase (CODH) catalyzes the bidirectional conversion of CO2 and CO at a heterometallic [NiFe4S4] cluster active site. A key component of the Wood-Ljungdahl pathway for the upconversion of C1 products, this enzymatic reaction offers a promising approach for reducing anthropogenic carbon emissions. However, CODH has proven difficult to study due to its high oxygen sensitivity and complex structure, highlighting the need for a simplified protein model system to elucidate its catalytic mechanism for CO2 reduction. Building upon our previous work reconstituting the native [Fe4S4] cluster with a [NiFe3S4] cluster in the Pyrococcus furiosus (Pf) wild-type ferredoxin (Fd), here we present D14C and D14H mutants of Pf Fd. The D14C mutant possesses a tetracysteinate primary coordination sphere that more closely resembles the active site of CODH, while the D14H mutant has a histidine residue coordinating to the Ni metal site to explore primary coordination sphere effects at the [NiFe3S4] cluster. With CODH catalyzing CO2 reduction at –530 mV, a fundamental goal for our models is to approach this potential. The electron-transfer properties of the variants containing a [NiFe3S4] cluster will be explored using protein-film electrochemistry across a range of pH and salt concentrations, as well as in the presence of relevant substrates, to assess the suitability of our models to mimic CODH electron transfer.

Volatile organic compounds (VOCs) are emitted into the atmosphere from a variety of anthropogenic and natural sources. Once emitted, they undergo oxidation. This plays a key role in important atmospheric processes including secondary organic aerosol and ozone formation. Water vapor is one of the most abundant minor constituents of the atmosphere, but its impact on VOC oxidation remains understudied, due in part to technical challenges with its introduction into atmospheric oxidation experiments. In previous work, there has been limited evidence that water may catalyze the unimolecular and self-reactions of peroxy radicals (RO2), which are a key intermediate in VOC oxidation. Here, we computationally examine interactions between water and RO2 derived from isoprene oxidation, which is well-understood under dry conditions, and is one of the most abundant non-methane VOC. More specifically, we focus on identifying conformers of isoprene-derived RO2-water complexes in order to accurately model the interaction between water and RO2. We employ a multi-step conformational sampling technique using ABCluster, CREST, and density functional theory calculations. The Jammy Key framework is used to interface between the various software. The results will provide insight into how water can affect oxidation mechanisms and kinetics of VOCs, and will inform the design and interpretation of environmental chamber experiments focused on probing the role of water in isoprene oxidation.