Complete Proteomic-Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic Flux and Lignin in Populus trichocarpa

Abstract

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a longstanding paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.

Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O -methyltransferase activity in poplar monolignol biosynthesis

“…S1B). PtrPAL4 and PtrPAL5 are xylem-specific and abundant and therefore are the key PALs involved in monolignol biosynthesis (12,23). PtrPAL1 is shoot-specific, suggesting its involvement in other pathways (23).…”

“…AldOMT (EC 2.1.1.68) catalyzes the S-adenosyl-L-methionine (SAM)-dependent methylation of 3-and 5-hydroxyl groups of precursors for syringyl monolignol biosynthesis (12). A total of 25 gene models encode putative AldOMTs in the genome of P. trichocarpa.…”

“…predicted necessary to modify metabolic-flux for syringyl monolignol biosynthesis (12). PtrAldOMT2 has the most abundant monolignol biosynthetic gene transcript in stem-differentiating xylem (SDX) of P. trichocarpa and is the third highest transcribed gene in the whole SDX transcriptome (7).…”

“…In the monolignol biosynthetic pathway, most enzymes are produced in excess of what is required for a normal lignin phenotype (12). This characteristic is particularly true for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to syringyl monolignol biosynthesis in Populus trichocarpa.…”

“…Monolignol biosynthesis is regulated by transcriptional activation and suppression by wood-associated NAC (NAM, ATAF1/2, and CUC2) domain factors and alternative splicing (5)(6)(7). At the metabolic level, monolignol biosynthesis is regulated by protein interactions (8,9) and a complex network of feed-forward and -back enzyme inhibitions (10)(11)(12)(13).…”