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...
In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.
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