Monolignol pathway 4-coumaric acid:coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes

Abstract

4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.

Figure 1.

The proposed monolignol biosynthetic pathway. The principal pathways for the biosynthesis of monolignols 4-coumaryl, coniferyl, and sinapyl alcohols are highlighted in gray. The first step in the pathway involves the deamination of Phe to cinnamic acid by phenylalanine ammonia-lyase (PAL). A multiple protein complex (C4H-C3H) consists of cinnamic acid 4-hydroxylase (C4H) and 4-coumarate 3-hydroxylase (C3H) hydroxylate cinnamic acid at the ring 4 position to 4-coumaric acid and subsequently at the ring-3 position of 4-coumaric acid to caffeic acid (Chen et al., 2011). 4CL then activates the hydroxycinnamic acids to their corresponding CoA thioesters. The C4H-C3H protein complex, in conjunction with 4-hydroxycinnamoyl-CoA:quinate shikimate 4-hydroxycinnamoyltransferase (HCT), hydroxylate 4-coumaroyl-CoA, at the 3 position to caffeoyl-CoA. Caffeoyl-CoA O-methyltransferase (CCoAOMT), coniferyl aldehyde 5-hydroxylase (CAld5H), and 5-hydroxyconiferaldehyde O-methyltransferase (AldOMT) modify the phenolic ring at the 3 and 5 positions through a series of hydroxylation and methoxylation reactions. Cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) reduce the phenolic intermediates to monolignols.

Figure 2.

Western blotting of native and recombinant Ptr4CL3 and Ptr4CL5 using phospho-specific antibodies and 4CL antibodies. A, Positive control: phosvitin. B, Negative control: soybean (Glycine max) trypsin inhibitor. C and D, Ptr4CL3 and Ptr4CL5 pulled down from P. trichocarpa SDX crude proteins using anti-aspen 4CL1 antibodies and probed with phospho-specific antibodies (C) and anti-aspen 4CL1 antibodies (D). E and G, Recombinant Ptr4CL3 proteins probed with anti-aspen 4CL1 antibodies (E) and phospho-specific antibodies (G). F and H, Recombinant Ptr4CL5 proteins probed with anti-aspen 4CL1 antibodies (F) and phospho-specific antibodies (H).

Figure 3.

Inhibition of Ptr4CL3-catalyzed 4-coumaric acid CoA ligation by caffeic acid. A, The double reciprocal plot shows that the mode of inhibition is pure competitive. Error bars represent the se of three technical replicates. B, K′_m/V′_max against [caffeic acid] is used to derive K_ic (Eq. 5).

Figure 4.

Inhibition of Ptr4CL5-catalyzed 4-coumaric acid CoA ligation by caffeic acid. A, The double reciprocal plot shows that the mode of inhibition is mixed competitive and uncompetitive. Error bars represent the se of three technical replicates. B, 1/V′_max against inhibitor concentration is used to derive the K_iu (Eq. 5). C, K′_m/V′_max against [caffeic acid] is used to derive K_ic (Eq. 5).

Figure 5.

Caffeic acid causes substrate self-inhibition of Ptr4CL5. A, Self-inhibition was not observed for Ptr4CL3 with increasing concentration of caffeic acid. B, Caffeic acid substrate self-inhibition of Ptr4CL5 when substrate concentration exceeds 55 µm. C, Secondary polynomial curve derived from the self-inhibition of Ptr4CL5 with caffeic as the substrate. D, The plot used to derive the K_is (Eq. 8). Error bars represent the se of three technical replicates.

Figure 6.

Absolute quantitation of the hydroxycinnamic acids in P. trichocarpa SDX extracts using stable isotope dilution-based LC-MS/MS. Error bars represent the se of 12 replicates (four biological replicates each with three technical replicates).

Figure 7.

Mathematical simulations for the reaction fluxes of Ptr4CL3 (A) and Ptr4CL5 (B) with individual substrates (4-coumaric, caffeic, or ferulic acid).

Figure 8.

Mathematical simulations and experimental validation of CoA ligation fluxes for Ptr4CL3 (A) and Ptr4CL5 (B) reactions in the presence of a mixture of 4-coumaric, caffeic, and ferulic acids. Error bars represent the se of three technical replicates.