4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes

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 x 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.

Figure 1

Native poplar H11 4CL isoforms. Proteins from developing secondary xylem (A), elicitor-treated cell culture (B), and young leaves (C) of clone H11 were extracted and separated by FPLC on an anion-exchange column. 4CL activity using 4-coumarate as a substrate was assayed in 1-mL fractions eluted using the nonlinear KCl gradient indicated. The fractions from each peak with the highest activities are noted in brackets. The substrate specificity of each isoform was tested using a pool of the fractions with highest activity (graphs on right), using hydroxycinnamate substrates at 0.2 mm. Error bars represent the sd of the average of three replicates. Asterisks indicate the absence of detectable activity. The average enzyme activities using 4-coumarate as a substrate, which were taken as 100%, are as follows: A, xylem peak I, 95.8 pkat/mL; xylem peak II, 153.0 pkat/mL; B, elicited cells, 166.0 pkat/mL; and C, young leaves peak I, 9.3 pkat/mL; young leaves peak II, 7.4 pkat/mL.

Figure 2

Restriction maps of poplar 4CL cDNA clones. RI, EcoRI; RV, EcoRV. Arrowheads represent placement of 4CL-specific primers (a and b) used to amplify a 670-bp H11 genomic DNA fragment to replace the 5′ end of 4CL-2, which contained an erroneous stop codon. The asterisk represents the approximate position of that stop codon.

Figure 3

Amino acid sequence comparison of 4CL-9, 4CL-216, and other known 4CL sequences. The full predicted amino acid sequence of 4CL-9 is given; amino acids in 4CL-216 are shown only where they differ from 4CL-9. Amino acids identical in all known 4CL sequences, including those of 4CL-216 and 4CL-9, are shown (IDE). A gap introduced to maximize the alignment is indicated by a dot in the 4CL-9 sequence. Conserved regions I (AMP-binding site) and II (putative catalytic site) are boxed; conserved Cys residues and the putative stop codons are indicated by asterisks.

Figure 4

Genomic Southern-blot analysis of 4CL genes in parental and hybrid poplar genotypes in a controlled cross. Genomic DNA (10 μg) from parental genotypes P. trichocarpa 93-968 and P. deltoides ILL129 and F₁ progeny 53-246 was cut with the four enzymes indicated and a Southern blot was prepared. The blot was hybridized sequentially to the cDNA probes indicated. X, XbaI; H, HindIII; R, EcoRV; and B, BamHI. Migration of size markers (in kilobases) is shown.

Figure 5

Northern-blot analysis of poplar 4CL mRNA levels. Total RNA (10 μg) from different tissues and organs was separated on duplicate formaldehyde agarose gels, transferred to nylon membranes, and hybridized to either 4CL-216 or 4CL-9 cDNA probes. ELI+, Elicitor-treated tissue culture cells; ELI−, untreated control cells. The membranes were stripped and rehybridized with an rRNA probe (pHA2) to demonstrate evenness of loading.

Figure 6

SDS-PAGE and immunoblot analysis of recombinant 4CL proteins. SDS-PAGE gel (A) and an immunoblot of a parallel gel (B) reacted with antiserum specific to parsley 4CL (Ragg et al., 1981). Crude extracts (20 μg) of Sf9 cells infected with 4CL-216 and 4CL-9 baculovirus constructs, and 4CL-216 and 4CL-9 (1 μg each) FPLC-purified from infected Sf9 cells, were loaded as shown. wt Sf9, Wild-type (uninfected) Sf9 cells. Crude poplar extracts were derived from elicitor-treated cells (E), differentiating xylem (X), and young leaves (YL). Molecular mass standards (in kilodaltons) are shown to the left.

Figure 7

Substrate-utilization profiles of recombinant 4CL. A, Recombinant 4CL-216; B, recombinant 4CL-9. 4CL enzyme activity (right-hand graphs) was measured using FPLC-purified 4CL-216 or 4CL-9 (left-hand graphs) and 0.2 mm concentrations of hydroxycinnamic acids. Results are averages of three trials; error bars represent sd values. Asterisks indicate the absence of detectable activity. Results are reported as a percentage of the activity against 4-coumaric acid, which was 32.0 pkat/μg protein for 4CL-216 and 119.2 pkat/μg protein for 4CL-9 in the experiment shown.