Enabling microbial syringol conversion through structure-guided protein engineering

Abstract

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.

Keywords: P450; biorefinery; demethylase; lignin.

Scheme 1.

O-demethylation of guaiacol to form catechol and formaldehyde (A) and syringol to form pyrogallol and two formaldehydes (B). The singly demethylated species, 3MC, is expected to form as an intermediate in reaction (B). See Fig. 1.

Fig. 1.

Quantitative analyses of substrate consumption and product generation indicate nearly complete coupling of NADH/O₂ consumption to substrate O-demethylation for guaiacol and progressively more uncoupling for syringol and 3MC. NADH (200 µM) and guaiacol, syringol, or 3MC (100 or 200 µM) were incubated in air with 0.2 μM GcoA-F169A and GcoB (each with 25 mM Hepes, 50 mM NaCl, pH 7.5 at 25 °C and 210 µM O₂). Reactants and products were quantified when the UV/vis spectrum ceased changing and the reaction was deemed complete. The total NADH consumed is compared with the amounts of formaldehyde and demethylated aromatic compound produced. Pyrogallol, the O-demethylated product of 3MC, is unstable in air under the conditions used in the assay and was not detected. Error bars represent ±1 SD from three or more independent measurements. P values comparing NADH consumption and formaldehyde production were 0.035 for guaiacol, 0.20 for 100 µM syringol, 0.41 for 200 µM syringol, and 0.0035 for 3MC. For NADH consumption and aromatic product production, these P values were 0.011 for guaiacol, 0.00031 for 100 µM syringol, and 0.0084 for 200 µM syringol.

Fig. 2.

Structure-guided active site engineering of GcoA. Superpositions of WT and GcoA-F169A ligand-bound structures of GcoA, the P450 monooxygenase component of GcoAB, are shown. The heme is colored in bronze stick. (A) The guaiacol (green) and syringol (pink) complexes with WT GcoA are shown with the position of the GcoA-F169 residue highlighted. The translation and rotation of syringol compared with guaiacol result in a shift of the target methoxy carbon away from the heme. The Fe(III) to guaiacol methoxy carbon distance is 3.9 Å, and the Fe(III) to proximal syringol methoxy carbon distance is 4.3 Å. Data from PDB ID: 5NCB (28) and 5OMU (28). (B) The engineered GcoA-F169A-syringol complex (blue) enables positioning of the reactive methoxy group relative to the heme in a mode consistent with productive guaiacol binding. GcoA-F169 from the guaiacol-bound WT structure is shown in green lines. (C) Superposition of the WT (green) and GcoA-F169A (yellow) guaiacol-bound complexes reveals that guaiacol sits in an identical position in both crystal structures. GcoA-F169 from the guaiacol-bound WT structure is shown in green lines.

Fig. 3.

GcoA-F169 in WT GcoA and the substrate access loop are significantly displaced with bound syringol. MD simulations with bound guaiacol indicate that GcoA-F169 (A) and the substrate access lid (B) are relatively stable (Movies S1 and S3). Introducing syringol results in increased flexibility of GcoA-F169 (C) and the substrate access loop (D) (Movies S2 and S4). In A–D, the position of each of the labeled Phe side chains (or, alternatively, the substrate access loop) is shown every 4 ns over the course of the 80-ns MD simulation. Substrate, the Phe side chains, and heme are shown in sticks, and the Fe atom and the O atom of a reactive heme-oxo intermediate are shown as spheres. Probability distributions are shown for the rmsd of the six ring carbons of GcoA-F169 from their crystal structure positions (E) and the reaction coordinates for opening/closing of the substrate access loop (as defined in SI Appendix; lower values indicate more open configurations) (F).

Fig. 4.

Bioinformatic analysis of CYP255A sequences indicates variability in the 169th sequence position. Conservation of residues within 6 Å of guaiacol in GcoA, determined via an analysis of protein sequences from 482 GcoA homologs. (Details provided in SI Appendix.) Conservation scores are reported as percentiles. F169 is less conserved than 73% of the positions in GcoA. Data from PDB ID: 5NCB (28).

Fig. 5.

GcoA-F169A converts syringol in vivo. (A) A pathway for in vivo syringol O-demethylation to pyrogallol and cleavage to 2-pyrone 6-carboxylate is proposed. (B) After 6 h, strains were analyzed for their ability to turn over syringol via ¹H NMR spectroscopy. Syringol (green) is completely converted to pyrogallol (pink), 3MC (blue), or 2-pyrone 6-carboxylate (purple) in AM157. The WT GcoA enzyme in AM156 shows only small amounts of conversion. AM155, which does not express GcoA, shows no conversion.