Modification of 4-coumarate:coa ligase proteins via mutagenesis and domain-swapping alters hydroxycinnamate substrate selectivity and phenylpropanoid metabolism in Populus
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4-coumarate:CoA ligase (4CL) activates hydroxycinnamates to their corresponding CoA esters for utilization in the biosynthesis of phenylpropanoid end products such as lignin and flavonoids. Multiple 4CL isoforms were found in all species examined, and these isoforms were grouped into three classes by phylogenetic analysis. Lignin biosynthesis-associated isoforms in angiosperms and gymnosperms segregated into Class I and Class III, respectively. Non-lignin phenylpropanoid biosynthesis-associated 4CLs of both angiosperms and gymnosperms clustered into Class II. This suggests that distinct 4CLs evolved before the divergence of spermatophytes. In vitro enzyme kinetic analysis revealed a correlation between 4CL phylogenetic classification and substrate selectivity. Homology modeling and site-directed mutagenesis identified residue 302 in the substrate binding pocket as a major determinant of 4CL substrate selectivity. Transgenic Populus over-expressing wild-type or mutant 4CLs with different substrate selectivity exhibited distinct phenylpropanoid phenotypes. Unexpectedly, little metabolic change was observed in the transgenic Populus over-expressing a loblolly pine Pta4CL1 or a Pta4CL1 domain-swap mutant. The weak transgenic effects were in contrast to the significant metabolic changes in transgenic Populus expressing loblolly pine Pta4CL3. One explanation for the distinct transgenic effects is that the backbone of Pta4CL1, which shares low sequence identity with Populus 4CLs, may be incompatible with the phenylpropanoid metabolons in Populus. Class II Pta4CL3, on the other hand, is evolutionary conserved with angiosperm 4CLs, and can exert an effect on Populus metabolism.