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

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

Authors:

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

Publication Date:

Thu Oct 01 00:00:00 EDT 2015

Research Org.:

Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Org.:

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

OSTI Identifier:

1760082

Alternate Identifier(s):

OSTI ID: 1235889

Report Number(s):

BNL-111732-2016-JA
Journal ID: ISSN 0021-9258; S0021925820495420; PII: S0021925820495420

Grant/Contract Number:  

DEAC0298CH10886; SC00112704

Resource Type:

Published Article

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Name: Journal of Biological Chemistry Journal Volume: 290 Journal Issue: 44; Journal ID: ISSN 0021-9258

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

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