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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 Enzymatic strategies for controlling and harnessing the oxidative power of O 2(Montana State University - Bozeman, College of Letters & Science, 2018) Machovina, Melodie M.; Chairperson, Graduate Committee: Jennifer DuBois; Robert J. Usselman and Jennifer L. DuBois were co-authors of the article, 'Monoxygenase substrates mimic flavin to catalyze cofactorless oxygenations' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Emerald S. Ellis, Thomas J. Carney, Fikile R. Brushett and Jennifer L. DuBois were co-authors of the article, 'Understanding how a cofactor-free protein environment lowers the barrier to O 2 reactivity' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Sam J. B. Mallinson, Rodrigo L. Silveira, Marc Garcia-Borras, Nathan Gallup were authors and Christopher W. Johnson, Mark D. Allen, Munir S. Skaf, Michael F. Crowley, Ellen L. Neidle, Kendall N. Houk, Gregg T. Beckham, Jennifer L. DuBois and John E. McGeehan were co-authors of the article, 'A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion' in the journal 'Nature Communications' which is contained within this dissertation.; Sam J.B. Mallinson was an author and Brandon C. Knott, Marc Garcia-Borras, Alexander W. Meyers, Lintao Bu, Japheth Gado, April Oliver, Graham P. Schmidt, J. Hinchen, Michael F. Crowley, Christopher W. Johnson, Ellen L. Neidle, Christina M. Payne, Gregg T. Beckham, Kendall N. Houk, John E. McGeehan and Jennifer L. DuBois were co-authors of the article, 'Enabling microbial syringol conversion through structure-guided protein engineering' submitted to the journal 'PNAS' which is contained within this dissertation.; Dissertation contains one article of which Melodie M. Machovina is not the main author.Dioxygen, one of Nature's most powerful oxidants, is essential for countless biological reactions. To harness this oxidant's power while minimizing toxicity, enzymes evolved to interact with O 2, activate it, and poise it for catalysis with substrates. This dissertation explores how two very different enzyme families, monooxygenases and a new class of cytochrome P450s, utilize this powerful oxidant. Previously, it was thought that cofactors are essential for O 2 activation; however, a subset of O 2-utilizing enzymes that catalyze direct reactions between substrate and O 2 was recently discovered, including nogalamycin monoxygenase (NMO). To probe how the protein environment affects thermodynamic and kinetic barriers of O 2 activation, we used a suite of techniques, including: UV/vis (transient and conventional) and electron paramagnetic resonance spectroscopies, O 2 consumption, high-performance liquid chromatography (HPLC), and cyclic voltammetry. Here, we provide evidence that the NMO mechanism has similar characteristics to that in flavoenzymes; in NMO, the substrate, acting in lieu of flavin, donates an electron to O 2, activating it to superoxide with the protein environment facilitating this by lowering the reorganization energy. The last half of this dissertation describes the discovery and engineering of a new class of cytochrome P450 enzymes that employ heme-iron oxygen activation to demethylate key lignin degradation products, forming central carbon intermediates that are precursors for bioplastics. The P450 GcoAB, comprised of the oxidase GcoA and the reductase GcoB, is efficient at demethylating G-lignin, but shows poor reactivity towards S-lignin. Using a structure-guided mutagenesis approach, we generated a variant, F169A GcoA, that is more efficient than wild-type at demethylating G-lignin and the only enzyme that efficiently degrades S-lignin. We characterized this variant, and the wildtype enzyme, using biochemical (UV/vis spectroscopy, HPLC), structural (X-ray crystallography), and computational (Molecular Dynamics and Density Functional Theory). Currently, we are testing the in vitro efficiency of additional variants evolved using a directed evolution approach. The results presented in the following chapters explore the mechanisms of several enzymes. Understanding how O2 is activated and utilized across diverse enzymatic systems provides valuable knowledge that can aid in future design and engineering of systems that use this 'green' oxidant, particularly for large-scale industrial applications.Item Enabling microbial syringol conversion through structure-guided protein engineering(2019-07-19) Machovina, Melodie M.; Mallinson, Sam J. B.; Knott, Brandon C.; Meyers, Alexander W.; Garcia-Borras, Marc; Bu, Lintao; Gado, Japheth E.; Oliver, April; Schmidt, Graham P.; Hinchen, Daniel J.; Crowley, Michael F.; Johnson, Christopher W.; Neidle, Ellen L.; Payne, Christina M.; Houk, Kendall N.; Beckham, Gregg T.; McGeehan, John E.; DuBois, Jennifer L.Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.Item How a cofactor-free protein environment lowers the barrier to O2 reactivity(2019-01) Machovina, Melodie M.; Ellis, Emerald S.; Carney, Thomas J.; Brushett, Fikile R.; DuBois, Jennifer L.Molecular oxygen (O2)-utilizing enzymes are among the most important in biology. The abundance of O2, its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. While most O2-dependent enzymes have an absolute requirement for an O2-activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O2 using only the protein environment. Nogalamycin monooxygenase (NMO) from Streptomyces nogalater is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O2. Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pKa by 1.0 unit (ΔG*= 1.4 kcal mol-1). We found that the one-electron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside NMO, corresponding to a ΔΔG⁰′ of 2.0 kcal mol-1 (0.087 eV) and that the activation barrier, ΔG‡, is lowered by 4.8 kcal mol-1 (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol-1 (1.4 eV) in the reorganization energy (l), which constitutes the major portion of the protein environment’s effect in lowering the reaction barrier. A similar role for the protein has been proposed in several cofactor-dependent systems and may reflect a broader trend in O2-utilizing proteins. In summary, NMO’s protein environment facilitates direct electron transfer, and NMO accelerates rate-limiting electron transfer by strongly lowering the reorganization energy.Item Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations(2016-08) Machovina, Melodie M.; Usselman, Robert J.; DuBois, Jennifer L.Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means.