A Scots pine 4-coumarate:CoA ligase with high cinnamic acid affinity is the primary candidate for pinosylvin biosynthesis

Pinosylvin and its derivatives are defense-related stilbenoids that contribute to pathogen and decay resistance of Scots pine (Pinus sylvestris L.) heartwood. While most enzymes in the pinosylvin biosynthesis pathway in Scots pine have been identified, the enzyme responsible for activating cinnamic acid to cinnamoyl-CoA, an essential precursor for pinosylvin biosynthesis, has remained unknown. In this study, we explored Scots pine transcriptomic data to observe the expression profiles of the 4-coumarate:CoA ligase (4CL) genes and produced the enzymes in Nicotiana benthamiana to assay their kinetic properties. We identified and characterized four 4CL isoforms, one of which (Ps4CL2) was both co-expressed with pinosylvin biosynthesis genes under stilbene-inducing conditions and exhibited an unusually high affinity for cinnamic acid surpassing that of previously characterized cinnamate-activating 4CLs. Through site-directed mutagenesis and domain-swapping experiments with the closely related Ps4CL3, we showed that substrate preference in Ps4CL2 results from multiple interacting regions within the enzyme. Our findings establish Ps4CL2 as the prime candidate for cinnamate activation as the last missing enzyme for pinosylvin biosynthesis in Scots pine and provide insight into enzyme specialization within the 4CL family.