Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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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 Coenzyme M biosynthesis in bacteria involves phosphate elimination by a functionally distinct member of the aspartase/fumarase superfamily(2018-04) Partovi, Sarah E.; Mus, Florence; Gutknecht, Andrew E.; Martinez, Hunter A.; Tripet, Brian P.; Lange, Bernd Markus; DuBois, Jennifer L.; Peters, John W.For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly to the PEP-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5\'-phosphate-containing enzyme with homology to D-cysteine desulfhydrases. Known AFS members catalyze beta-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes beta-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing beta-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate beta-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.Item Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b(2018-03) Streit, Bennett R.; Celis Luna, Arianna I.; Moraski, Garrett C.; Shisler, Krista A.; Shepard, Eric M.; Rodgers, Kenton R.; Lukat-Rodgers, Gudrun S.; DuBois, Jennifer L.The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 forms a radical species within 30 sec of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 sec, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 sec after initiating the reaction with H2O2, nor did it change the ~270 sec required for the radical signal to recede to ≤ 10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145 was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ~2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cbeta-H bond was partly rate limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cbeta-H to Tyr-145.Item Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation(2017-08) Mus, Florence; Eilers, Brian J.; Alleman, Alexander B.; Kabasakal, Burak V.; Wells, Jennifer N.; Murray, James W.; Nocek, Boguslaw P.; DuBois, Jennifer L.; Peters, John W.Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO2) or its hydrated form, bicarbonate (HCO3 −), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO3 − to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ)2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform C-C bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO2 into biomass, a process of great significance to the global carbon cycle.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.