Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.

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.

How Chemical Environment Activates Anthralin and Molecular Oxygen for Direct Reaction.

The role of the chemical environment in promoting anthralin/O2 reactions was discovered using neat solvents to model the amino acids of a cofactor-independent oxygenase. Experimental and computational results highlight the importance of the substrate-enolate, which is accessed via energetically small, escalating steps in which the ground-state keto-isomer is tautomerized to an enol and then ionized by solvent. The resulting ion-pair is poised for spontaneous electron transfer to O2. Similar activation may be exploited in biological/nonbiological oxidations involving O2.

How a cofactor-free protein environment lowers the barrier to O2 reactivity.

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. Although 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 (λ), 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.