4-Coumarate:Coenzyme A Ligase in Hybrid Poplar¹
Properties of Native Enzymes, cDNA Cloning, and Analysis of Recombinant Enzymes

Sandra M. Allina², Aviva Pri-Hadash², David A. Theilmann, Brian E. Ellis, and Carl J. Douglas^*

Departments of Botany (S.M.A., A.P.-H., C.J.D.), and Plant Science (A.P.-H., D.A.T., B.E.E.), University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; and University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada V0H 1Z0 (D.A.T.)

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.

Plant Physiol. (1998) 116: 743-754
Copyright Clearance Center:   0032-0889/98/116/0743/12
© 1998 American Society of Plant Physiologists

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