4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1

Abstract

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa ؋ Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.The enzyme 4CL (EC 6.2.1.12) catalyzes the formation of CoA thioesters of hydroxycinnamic acids by a two-step reaction mechanism that involves the hydrolysis of ATP (Gross and Zenk, 1974). These thioesters serve as substrates for a number of important reactions within plant phenylpropanoid metabolism. Depending on the species and tissue, formation of hydroxycinnamate esters and amides often involves CoA derivatives of 4-coumaric, caffeic, and/or ferulic acid, whereas flavonoid and stilbene biosynthesis requires cinnamoyl and/or 4-coumaroyl CoA esters as the substrates (Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995;Holton and Cornish, 1995). The biosynthesis of salicylic acid from cinnamic acid may occur via oxidation of a cinnamoyl-CoA intermediate (Ryals et al., 1996), and within virtually all higher plants, generation of lignin monomers is presumed to require the conversion of 4-coumaric acid, ferulic acid, and sinapic acid to the corresponding CoA esters in preparation for side-chain reduction .In keeping with this metabolic demand for different hydroxycinnamoyl CoA esters, 4CL preparations from plants are normally found to be able to use a number of hydroxycinnamic acid derivatives as substrates (Gross and Zenk, 1974; Hahlbrock, 1975, 1977;Grand et al., 1983;Lozoya et al., 1988;Voo et al., 1995;Lee and Douglas, 1996). It has also been proposed, however, that different isoforms of 4CL might possess different patterns of substrate preference, and characterization of 4CL isoforms from some plants supports this (Knobloch and Hahlbrock, 1975;Ranjeva et al., 1976;Wallis and Rhodes, 1977;Grand et al., 1983). Such diversity could conceivably enable particular 4CL isoforms to efficiently supply an appro...

Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium Streptomyces coelicolor A3(2)

“…ScCCL had 44% identity and 58% similarity to A. thaliana 4CL2. Among the 4CL enzymes, two highly conserved peptide motifs, box I (SSGTTGLPKGV) and box II (GEICIRG), have been identified (1,2,10,11,18,26,28). In the ScCCL sequence, both motifs were also highly conserved.…”

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

“…4), all of which exhibit a large structural, enzyme-kinetic, and apparent evolutionary diversity. The very opposite is exemplified by the much smaller and narrowly confined St4CL, Pc4CL, and Nt4CL families, which appear to consist of no more than two (St4CL) or three (Pc4CL, Nt4CL) nearly sequence-identical isoenzymes with indistinguishable substrate specificities (6,7,30). However, in all of these cases, with the single exception of At4CL, the true family sizes remain unknown until fully sequenced genomes are available.…”

“…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).…”

“…As far as analyzed, 4CL has been shown to occur in the form of multiple isoenzymes, which frequently (1-5), but not always (6,7), exhibit distinct substrate affinities that appear to coincide with specific metabolic functions. Of the four naturally occurring, potential substrates illustrated in Fig.…”

<i>In silico</i> Analysis of 4CL Family in <i>Scutellaria baicalensis</i> through Biocomputational Tools and Servers

“…So 4CL is one key enzyme of flavonodids biosynthesis pathway (Fan et al, 2007). Many studies revealed that 4cl gene was a multigene family: Two 4cl genes are cloned from Scutellaria baicalensis Georgi and three 4cl genes are isolated and characterized in Hyvrid Poplar (Allina et al, 1998). With further 4CL enzymological identification, genetic mutation and crystal modeling, the studies on the structure and evolution were implemented extensively (Cukovic et al, 2001;Schneider et al, 2003) and then some highly conserved enzyme active sites residues were revealed, such as Box I (SSGTTGLPKGV) and Box II (GEICIRG) (Stuible and Kombrink, 2001), sbd I (N-terminal domain) and abd II (C-terminal domain) .…”