| Abstract: | As a step toward predictive modeling of flux through the pathway of monolignolbiosynthesis in stem differentiating xylem of Populus trichocarpa,we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro toform a heterotetrameric protein complex. This conclusion is based on lasermicrodissection, coimmunoprecipitation, chemical cross-linking, bimolecularfluorescence complementation, and mass spectrometry. The tetramer is composed ofthree subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. Thisprotein–protein interaction affects the direction and rate of metabolic fluxfor monolignol biosynthesis in P. trichocarpa. A mathematical modelwas developed for the behavior of 4CL3 and 4CL5 individually and in mixtures thatform the enzyme complex. The model incorporates effects of mixtures of multiplehydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition,and self-inhibition, along with characteristic of the substrates, the enzymeisoforms, and the tetrameric complex. Kinetic analysis of different ratios of theenzyme isoforms shows both inhibition and activation components, which are explainedby the mathematical model and provide insight into the regulation of metabolic fluxfor monolignol biosynthesis by protein complex formation. |
