Browsing by Author "Streit, Bennett R."

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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 â‰¤ 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.
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.