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

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

doi:10.1074/jbc.M115.684217

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

Journal Article · Thu Oct 01 00:00:00 EDT 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M115.684217· OSTI ID:1760082

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

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. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: DEAC0298CH10886; SC00112704

OSTI ID:: 1760082

Alternate ID(s):: OSTI ID: 1235889

Report Number(s):: BNL-111732-2016-JA; S0021925820495420; PII: S0021925820495420

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Vol. 290 Journal Issue: 44; ISSN 0021-9258

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 9 works

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59 BASIC BIOLOGICAL SCIENCES
monolignol
4-O-methyltransferase
lignin
crystal structure
directed evolution
mutagenesis
plant cell wall
protein engineering

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

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