Volume 8, Issue 1, Pages 176-187 (January 2015) 4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit 4-Coumaric Acid:Coenzyme A Ligases and Modulate Metabolic Flux for 3-Hydroxylation in Monolignol Biosynthesis of Populus trichocarpa  Chien-Yuan Lin, Jack P. Wang, Quanzi Li, Hsi-Chuan Chen, Jie Liu, Philip Loziuk, Jina Song, Cranos Williams, David C. Muddiman, Ronald R. Sederoff, Vincent L. Chiang  Molecular Plant  Volume 8, Issue 1, Pages 176-187 (January 2015) DOI: 10.1016/j.molp.2014.12.003 Copyright © 2015 The Author Terms and Conditions

Figure 1 A Section of the P. trichocarpa Monolignol Biosynthetic Pathway. Starting from cinnamic acid, all metabolites in this section of the monolignol biosynthetic pathway are numbered and underlined. The cofactors are also depicted in each reaction in gray. Reaction A is the formation of 4-coumaroyl-CoA from 4-coumaric acid and reaction B is the formation of caffeoyl-CoA from caffeic acid. CSE (dotted line), a recently discovered enzyme in Arabidopsis, converts caffeoyl shikimic acid to caffeic acid. H, G, and S refer to the type of subunits in lignin, 4-hydroxyphenyl, guaiacyl, and syringyl, respectively. Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 2 4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit Recombinant Ptr4CL3 and Ptr4CL5 Activities. (A) Inhibition by shikimic acid esters of Ptr4CL3 activity for 4-coumaroyl-CoA production. (B) Inhibition by shikimic acid esters of Ptr4CL3 activity for caffeoyl-CoA production. (C) Inhibition by shikimic acid esters of Ptr4CL5 activity for 4-coumaroyl-CoA production. (D) Inhibition by shikimic acid esters of Ptr4CL5 activity for caffeoyl-CoA production. The concentration of all substrates and inhibitors was 50 μM. The error bars represent one standard error of three technical replicates. Statistical testing was performed using the Student t-test (*p < 0.05; **p < 0.01). Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 3 Inhibition Kinetics Show that 4-Coumaroyl and Caffeoyl Shikimic Acids Have Distinct Effects on Ptr4CL3 and Ptr4CL5 Reactions. (A) Inhibition kinetics of Ptr4CL3 activity with 4-coumaric acid as the substrate and 4-coumaroyl shikimic acid as the inhibitor. (B) Inhibition kinetics of Ptr4CL5 activity with 4-coumaric acid as the substrate and 4-coumaroyl shikimic acid as the inhibitor. (C) Inhibition kinetics of Ptr4CL3 with caffeic acid as the substrate and 4-coumaroyl shikimic acid as the inhibitor. (D) Inhibition kinetics of Ptr4CL5 activity with caffeic acid as the substrate and 4-coumaroyl shikimic acid as the inhibitor. (E) Inhibition kinetics of Ptr4CL3 activity with 4-coumaric acid as the substrate and caffeoyl shikimic acid as the inhibitor. (F) Inhibition kinetics of Ptr4CL5 activity with 4-coumaric acid as the substrate and caffeoyl shikimic acid as the inhibitor. The plots of K′m/V′max are used to determine the Kic values and the Kiu values are derived from 1/V′max plots. Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 4 4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit Ptr4CL Reactions in SDX Extracts of P. trichocarpa. (A) Inhibition by 4-coumaroyl and caffeoyl shikimic acids on the formation of 4-coumaroyl-CoA in SDX extracts of P. trichocarpa. (B) Inhibition by 4-coumaroyl and caffeoyl shikimic acids on the formation of caffeoyl-CoA in SDX extracts of P. trichocarpa. The concentration of all substrates and inhibitors was 50 μM. The error bars represent one standard error of three technical replicates. Statistical testing was performed using the Student t-test (*p < 0.05; **p < 0.01). Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 5 4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit Ptr4CL Reactions in a Mixture of the Recombinant Proteins in a 3 to 1 Ratio of Ptr4CL3 and Ptr4CL5, Where Shikimic Acid Has No Inhibitory Effect. (A) Inhibition by 4-coumaroyl and caffeoyl shikimic acids on the formation of 4-coumaroyl-CoA, but not by shikimic acid. (B) Inhibition by 4-coumaroyl and caffeoyl shikimic acids on the formation of caffeoyl-CoA, but not by shikimic acid. The concentration of all substrates and inhibitors was 50 μM. The error bars represent one standard error of three technical replicates. Statistical testing was performed using the Student t-test (*p < 0.05; **p < 0.01). Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 6 Quantification of Transcript and Metabolite Levels of 4-Coumaroyl Shikimic Acid and 4-Coumaric Acid in PtrC3H3 Downregulated Transgenic and Wild-type of P. trichocarpa. (A) qRT-PCR result shows the reduced level of PtrC3H3 transcript in transgenic compared with wild-type. (B) Absolute protein quantification of PtrC3H3 in wild-type and transgenic plants. (C) Absolute quantification of 4-coumaroyl shikimic acid. (D) Absolute quantification of 4-coumaric acid. (E) Absolute quantification of caffeoyl shikimic acid. The error bars represent one standard error of three technical replicates. Statistical testing was performed using the Student t-test (**p < 0.01). Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions

Figure 7 The Equations from Chen et al. (2014) have been Modified to Include the Effects of Inhibition by 4-Coumaroyl and Caffeoyl Shikimic Acids on Ptr4CL Reactions Using 4-Coumaric Acid as the Substrate (A) and Caffeic Acid as the Substrate (B). These equations allow us to calculate reaction rates at different concentrations of substrates and inhibitors. Abbreviations: Km1 and Km2, the Michaelis-Menten constants for Ptr4CL3 and Ptr4CL5 respectively; kcat1 and kcat2, the turnover constants for Ptr4CL3 and Ptr4CL5 respectively; [E1t] and [E2t], the concentrations of Ptr4CL3 and Ptr4CL5, respectively (Shuford et al., 2012; Wang et al., 2014); [I1] and [I2], the concentrations of 4-coumaroyl shikimic acid and caffeoyl shikimic acid respectively; [I3] and [I4], the concentrations of caffeic and ferulic acids for reaction A, and 4-coumaric and ferulic acids for reaction B, respectively; K3ic1, K3iu1, K5ic1, and K5iu1, the competitive and uncompetitive inhibition constants for Ptr4CL3 and Ptr4CL5 when 4-coumaroyl shikimic acid is the inhibitor; K3ic2, K3iu2, K5ic2, and K5iu2, the competitive and uncompetitive inhibition constants for Ptr4CL3 and Ptr4CL5 when caffeoyl shikimic acid is the inhibitor; K3ic3, K3iu3, K5ic3, and K5iu3, the competitive and uncompetitive inhibition constants for Ptr4CL3 and Ptr4CL5 when caffeic acid is the inhibitor (reaction A) and 4-coumaric acid is the inhibitor (reaction B); K3ic4, K3iu4, K5ic4, and K5iu4, the competitive and uncompetitive inhibition constants for Ptr4CL3 and Ptr4CL5 when ferulic acid is the inhibitor; K5is, the substrate self-inhibition constant for Ptr4CL5 when caffeic acid is the substrate (Chen et al., 2013); k1, k2, and γ, the optimized parameters for Ptr4CL protein complex formation and regulation (Chen et al., 2014). Molecular Plant 2015 8, 176-187DOI: (10.1016/j.molp.2014.12.003) Copyright © 2015 The Author Terms and Conditions