Primary structures and catalytic properties of isoenzymes encoded by the two 4‐coumarate: CoA ligase genes in parsley

Abstract

We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.One active response of plants to environmental stress is the accumulation of secondary metabolites. Many of these plant metabolites are phenylpropanoid derivatives, e.g. flavonoids, isoflavonoids, coumarins, lignin and related phenolic compounds [I, 21. The CoA esters of 4-coumaric and other substituted cinnamic acids are essential intermediates in the biosynthesis of most or all of these compounds. The sequence of reactions leading to the formation of the thiol esters is called general phenylpropanoid metabolism [l] ( Fig.

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1

“…It is possible that sinapoyl-CoA in poplar and other plants is made via an alternative pathway involving hydroxylation and methoxylation of the CoA esters of hydroxycinnamic acids (Ye at al., 1994). The absence of activity against sinapic acid, coupled with the apparent absence of catalytically distinct 4CL isoforms in poplar and other plants (Lozoya et al, 1988;Voo et al, 1995;Lee and Douglas, 1996), makes it unlikely that the differential expression of 4CL gene family members encoding enzymes with different substrateutilization profiles is a mechanism used in poplar, or most other plants, to partition carbon into guaiacyl and syringyl lignin, or into other phenylpropanoid end products. However, there is evidence from tobacco (Lee and Douglas, 1996), pine (Zhang and Chiang, 1997), and now poplar that modification of 4CL enzyme activity, resulting in changes in its substrate-utilization profile, could be important in carbon partitioning.…”

“…However, our data show that 4CL isoforms that directly produce sinapoyl-CoA are unlikely to exist in poplar. Based on kinetic analysis of 4CL-9 and the substrateutilization profiles of 4CL-216, 4CL-9, and native poplar 4CL isoforms, poplar 4CL enzymes appear to be typical of those described for many plants (Wallis and Rhodes, 1977;Lozoya et al, 1988;Voo et al, 1995;Lee and Douglas, 1996) with activity against 4-coumaric acid Ͼ ferulic acid Ͼ caffeic acid, and no activity against sinapic acid. It is possible that sinapoyl-CoA in poplar and other plants is made via an alternative pathway involving hydroxylation and methoxylation of the CoA esters of hydroxycinnamic acids (Ye at al., 1994).…”

“…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 appropriate mixture of substrate(s) for specific metabolic sequences. However, in other plants in which they have been purified, no evidence for such catalytically distinct 4CL isoforms has emerged (Lozoya et al, 1988;Voo et al, 1995), and two divergent tobacco 4CL enzymes produced in recombinant form in Escherichia coli showed no differences in their substrate-utilization profiles (Lee and Douglas, 1996). Poplar (Populus spp.)…”

“…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).…”