Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

Yuanheng Cai,Mohammad-Wadud Bhuiya,John Shanklin,Chang-Jun Liu

doi:10.1074/jbc.m115.684217

Yuanheng Cai, Mohammad-Wadud Bhuiya + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m115.684217

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Abstract

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.

Highlights

Guaiacyl lignin dominates the polymer’s condensation, negatively affecting plant cell wall’s digestibility
There are three major monolignols, namely p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that differ in the number of methoxy groups on their aromatic rings and that lead to correspondingly three distinct structural subunits, the hydroxylphenyl (H),3 guaiacyl (G), and sinapyl (S) units when they are incorporated into lignin polymer (Fig. 1A)
Further optimizing MOMT3 activity via iterative saturation mutagenesis generated a set of tetra- and penta-mutant variants that includes the one carrying two additional substituted amino acids at its substrate binding pocket, i.e. F166W and H169F, designated MOMT5 (T133L/E165I/F166W/ F175I/H169F)

Summary

Background

Guaiacyl lignin dominates the polymer’s condensation, negatively affecting plant cell wall’s digestibility. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and the degradability of plant cell walls. We test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that methylates the condensed guaiacyl lignin precursor coniferyl alcohol. There are three major monolignols, namely p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that differ in the number of methoxy groups on their aromatic rings and that lead to correspondingly three distinct structural subunits, the hydroxylphenyl (H), guaiacyl (G), and sinapyl (S) units when they are incorporated into lignin polymer (Fig. 1A). The composition of the monolignol monomers determines the degree of lignin condensation, the complexity of the lignin structure, and thereby the degradability of plant cell walls [4]. The high S/G ratio of lignin is often associated with increased pulping yields [5, 6] and enzymatic sugar release [7], whereas a high content of G-units is associated with poor lignin degradation due to the presence of C-C bonds at the free C5 position [8]

The abbreviations used are

Results

Discussion