Chemistry & Biochemistry
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The Department of Chemistry and Biochemistry offers research-oriented programs culminating in the Doctor of Philosophy degree. The faculty in the department have expertise over a broad range of specialty areas including synthesis, structure, spectroscopy, and mechanism. In each of these fields, the strength of the department has been recognized at the international level.
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Item Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system(Wiley, 2024-05) Lusty Beech, Jessica; Maurya, Anjani K.; Rodrigues da Silva, Ronivaldo; Akpoto, Emmanuel; Asundi, Arun; Fecko, Julia Ann; Yennawar, Neela H.; Sarangi, Ritimukta; Tassone, Christopher; Weiss, Thomas M.; DuBois, Jennifer L.Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3β3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol−1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β-β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.Item Bacteroides thetaiotaomicron, a Model Gastrointestinal Tract Species, Prefers Heme as an Iron Source, Yields Protoporphyrin IX as a Product, and Acts as a Heme Reservoir(Bacteroides thetaiotaomicron, a Model Gastrointestinal Tract Species, Prefers Heme as an Iron Source, Yields Protoporphyrin IX as a Product, and Acts as a Heme Reservoir, 2023-03) Meslé, Margaux M.; Gray, Chase R.; Dlakić, Mensur; DuBois, Jennifer L.Members of the phylum Bacteroidetes are abundant in healthy gastrointestinal (GI) tract flora. Bacteroides thetaiotaomicron is a commensal heme auxotroph and representative of this group. Bacteroidetes are sensitive to host dietary iron restriction but proliferate in heme-rich environments that are also associated with colon cancer. We hypothesized that B. thetaiotaomicron may act as a host reservoir for iron and/or heme. In this study, we defined growth-promoting quantities of iron for B. thetaiotaomicron. B. thetaiotaomicron preferentially consumed and hyperaccumulated iron in the form of heme when presented both heme and nonheme iron sources in excess of its growth needs, leading to an estimated 3.6 to 8.4 mg iron in a model GI tract microbiome consisting solely of B. thetaiotaomicron. Protoporphyrin IX was identified as an organic coproduct of heme metabolism, consistent with anaerobic removal of iron from the heme leaving the intact tetrapyrrole as the observed product. Notably, no predicted or discernible pathway for protoporphyrin IX generation exists in B. thetaiotaomicron. Heme metabolism in congeners of B. thetaiotaomicron has previously been associated with the 6-gene hmu operon, based on genetic studies. A bioinformatics survey demonstrated that the intact operon is widespread in but confined to members of the Bacteroidetes phylum and ubiquitous in healthy human GI tract flora. Anaerobic heme metabolism by commensal Bacteroidetes via hmu is likely a major contributor to human host metabolism of the heme from dietary red meat and a driver for the selective growth of these species in the GI tract consortium.Item Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes(American Chemical Society, 2021-03) Ellis, Emerald S.; Hinchen, Daniel J.; Bleem, Alissa; Bu, Lintao; Mallinson, Sam J. B.; Allen, Mark D.; Streit, Bennett R.; Machovina, Melodie M.; Doolin, Quinlan V.; Michener, William E.; Johnson, Christopher W.; Knott, Brandon C.; Beckham, Gregg T.; McGeehan, John E.; DuBois, Jennifer L.Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.Item Pathways of Iron and Sulfur Acquisition, Cofactor Assembly, Destination, and Storage in Diverse Archaeal Methanogens and Alkanotrophs(2021-08) Johnson, Christina; England, Alexis; Munro-Ehrlich, Mason; Colman, Daniel R.; DuBois, Jennifer L.; Boyd, Eric S.Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F430) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved.