Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate

Saunia Withers,Fachuang Lu,Hoon Kim,Yimin Zhu,John Ralph,Curtis G Wilkerson

doi:10.1074/jbc.m111.284497

Abstract

Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be "monomer" precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters.

Highlights

In grasses, monolignols can be enzymatically preacylated and incorporated into lignin with unknown effects on lignin function
Gene Synthesis—OsPMT2 from O. sativa was submitted to Blue Heron Bio (State) for Escherichia coli codon optimization, synthesis, and cloning into the entry vector pENTR221 (Invitrogen)
Because p-coumaroylation is a distinctive feature of grass lignins, we reasoned that a grass specific HxxxD acyltransferase that is co-expressed with genes involved in monolignol biosynthesis would be a good candidate for the enzyme responsible for acylation of monolignols

Summary

Background

Monolignols can be enzymatically preacylated and incorporated into lignin with unknown effects on lignin function. Studies indicate that monolignols can be enzymatically preacylated by various acids including: acetates, which occur at low levels in hardwoods but at high levels in palms, kenaf, abaca, and sisal (6, 9 –11); p-hydroxybenzoates in palms and Populus species (willow, aspen, poplar) [12,13,14,15,16,17,18]; and p-coumarates in both C3 and C4 grasses (19 –22), prior to their incorporation into lignin The function of such acylation is essentially unknown, but these monolignol conjugates can be “monomer” precursors of lignin [23]. Identifying the enzyme responsible for acylating grass lignins will help us to determine the function of this abundant modification

EXPERIMENTAL PROCEDURES

The abbreviations used are

RESULTS

DISCUSSION