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Cloning and expression of genes encoding divergent 4-coumarate : CoA ligase in poplar Cukovic, Daniela

Abstract

The enzyme 4-coumarate:Coenzyme A ligase catalyzes the third and final step in the general phenylpropanoid metabolic pathway, providing activated thioester substrates for different phenylpropanoid products. To clone putative divergent members of the poplar 4CL gene family, degenerate primers directed to a part of the first exon, containing a putative AMP-binding motif, were used. Three new 4CL-like classes were distinguished among 72 cloned PCR amplification products. The new 4CL-like sequence classes, arbitrarily named 4CL6, 4CL10 and 4CL14, shared 61% to 96% deduced amino acid sequence identity over the 600 bp region compared. A full-length cDNA clone of 4CL6 was isolated by screening a xylem cDNA library with the PCR fragment. Isolation of a full-length cDNA clone of 4CL10 involved both screening a young leaf cDNA library, and 5' and 3' RACE. Sequence analysis showed that the most divergent member of the poplar 4CL gene family is 4CL10 (61% -65% amino acid identity with others), while 4CL6 is moderately divergent to previously isolated members. RNA blot analysis showed that the 4CL6 gene is expressed exclusively in xylem, green stem and root, while 4CL10 expression is restricted to young leaf, old leaf and root. Recombinant enzymes corresponding to the 4CL6 and 4CL10 genes were expressed using a baculovirus system. The recombinant 4CL6 was partially purified by FPLC, and showed a substrate utilization profile (4-coumaric acid > ferulic acid > caffeic acid > cinnamic acid, and no activity toward sinapic acid) similar to the profiles of the previously studied native and recombinant 4CL proteins.

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