Differential Substrate Inhibition Couples Kinetically Distinct 4-Coumarate:Coenzyme A Ligases with Spatially Distinct Metabolic Roles in Quaking Aspen

Abstract

4-Coumarate:coenzyme A ligase (4CL) activates hydroxycinnamates for entry into phenylpropanoid branchways that support various metabolic activities, including lignification and flavonoid biosynthesis. However, it is not clear whether and how 4CL proteins with their broad substrate specificities fulfill the specific hydroxycinnamate requirements of the branchways they supply. Two tissue-specific 4CLs, Pt4CL1 and Pt4CL2, have previously been cloned from quaking aspen (Populus tremuloides Michx.), but whether they are catalytically adapted for the distinctive metabolic roles they are thought to support is not apparent from published biochemical data. Therefore, single-and mixed-substrate assays were conducted to determine whether the 4CLs from aspen exhibit clear catalytic identities under certain metabolic circumstances. Recombinant Pt4CL1 and Pt4CL2 exhibited the expected preference for p-coumarate in single-substrate assays, but strong competitive inhibition favored utilization of caffeate and p-coumarate, respectively, in mixed-substrate assays. The Pt4CL1 product, caffeoyl-CoA, predominated in mixed-substrate assays with xylem extract, and this was consistent with the near absence of Pt4CL2 expression in xylem tissue as determined by in situ hybridization. It is interesting that the Pt4CL2 product p-coumaroyl-CoA predominated in assays with developing leaf extract, although in situ hybridization revealed that both genes were coexpressed. The xylem extract and recombinant 4CL1 data allow us to advance a mechanism by which 4CL1 can selectively utilize caffeate for the support of monolignol biosynthesis in maturing xylem and phloem fibers. Loblolly pine (Pinus taeda), in contrast, possesses a single 4CL protein exhibiting broad substrate specificity in mixed-substrate assays. We discuss these 4CL differences in terms of the contrasts in lignification between angiosperm trees and their gymnosperm progenitors. 4-Coumarate:coenzyme A (CoA) ligase (4CL) mediates activation of hydroxycinnamic acids 4-(p)-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA), 5-hydroxyferulic acid (5HFA), and sinapic acid (SA) into the high-energy intermediates used for biosynthesis of lignin, flavonoids, and various other protective, attractant, and signaling metabolites (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995;Higuchi, 1997;Whetten et al., 1998). Multiple 4CL isoforms with differential in vitro substrate specificities have been reported in several species (Knobloch and Hahlbrock, 1975;Ehlting et al., 1999), including aspen (Populus tremuloides Michx.; Hu et al., 1998), and these isoforms have been proposed to control the relative abundance of flavonoids and various lignin precursors (monolignols) during structural, protective, and reproductive development (Ranjeva et al., 1976;Knobloch and Hahlbrock, 1977;Grand et al., 1983). Wounding, UV light, and elicitors increase transcript abundance of different 4CL isoforms (Uhlmann and Ebel, 1993;Ehlting et al., 1999), reinforcing the model that there are distinct associations be...

Monolignol Pathway 4-Coumaric Acid:Coenzyme A Ligases in Populus. trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme A Ligation Fluxes

“…S3J), with a K ic value of 3.15 6 0.26 mM (Table IV). This K ic value is consistent with those obtained for recombinant protein 4CL1 (Harding et al, 2002), the aspen ortholog of PtrCL3. As verified by linear inhibition plots, all inhibitors tested here caused pure-competitive inhibition of Ptr4CL3 reactions, with distinct K ic values (Table IV; Supplemental Fig.…”

“…Ptr4CL3 efficiently converts all but sinapic acid into their corresponding CoA thioesters. The peptide sequence and particularly the biochemical functions of Ptr4CL3 demonstrate that Ptr4CL3 is a structural and functional ortholog of aspen 4CL1 previously identified and characterized (Hu et al, 1998(Hu et al, , 1999Harding et al, 2002). Ptr4CL5 mediates the CoA ligation of all five acids, and the functions of this 4CL have not been previously identified.…”

“…The 4CL gene product catalyzes CoA ligation of many hydroxycinnamic acids (Fig. 1) into the corresponding CoA thioesters for the biosynthesis of phenylpropanoids, such as monolignols (Walton and Butt, 1970;Knobloch and Hahlbrock, 1975;Higuchi, 1997;Lee et al, 1997;Hu et al, 1998Hu et al, , 1999Ehlting et al, 1999;Harding et al, 2002). Biochemical and genetic studies further demonstrated that a key function of monolignol 4CLs is the regulation of CoA ligation fluxes to affect the total lignin content in secondary cell walls in tobacco (Nicotiana tabacum; Kajita et al, 1996), Populus spp.…”

The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes

“…Considering the complexity and the extent of overlapping mRNA expression patterns that have been observed in various species for nearly all tested 4CL isoenzyme combinations (4,7,32,33), including At4CL1-3 (18), the pivotal position of 4CL in plant phenylpropanoid metabolism is probably reflected by a highly diversified metabolic grid (34)(35)(36). Moreover, the combination of distinct substrate preferences with differences in gene-promoter and protein fine structure may facilitate not only a large diversity of genetically programmed metabolic functions of the individual isoenzymes, including pathway-specific associations with biosynthetically related enzymes in metabolite channeling (37-39), but also regular or potential functional overlaps or mutual replacements upon malfunction or down-regulation, as has been investigated so far only for the class I isoforms (4,40,41).…”

Combinatorial modification of multiple lignin traits in trees through multigene cotransformation

“…Aspen xylem-specific promoter (Pt4CL1P)-GUS binary plasmid DNA (22) was used as a module for preparing the genetic constructs. The GUS fragment was replaced with either an antisense Pt4CL1 or a sense LsCAld5H cDNA with respect to the Pt4CL1P promoter.…”