Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.
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 isO-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 syringolO-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 inPseudomonas putidaKT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromaticO-demethylases in the biological conversion of lignin-derived aromatic compounds.
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.
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