Abstract
4-Coumarate: coenzyme A ligase (4CL) is a key enzyme involved in the early steps of the monolignol biosynthetic pathway. It is hypothesized to modulate S and G monolignol content in the plant. Lignin removal is imperative to the paper industry and higher S/G ratio governs better extractability of lignin and economics of the pulping process. This background prompted us to predict 3D structure of two isoforms of 4CL in Leucaena leucocephala and evaluate their substrate preferences. The 3D structure of Ll4CL1 and Ll4CL2 protein were created by homology modeling and further refined by loop refinement. Molecular docking studies suggested differential substrate preferences of both the isoforms. Ll4CL1 preferred sinapic acid (- 4.91 kcal/mole), ferulic acid (- 4.84 kcal/mole), hydroxyferulic acid (- 4.72 kcal/mole), and caffeic acid (- 4.71 kcal/mole), in their decreasing order. Similarly, Ll4CL2 preferred caffeic acid (- 6.56 kcal/mole, 4 H bonds), hydroxyferulic acid (- 6.56 kcal/mole, 3 H bonds), and ferulic acid (- 6.32 kcal/mole) and sinapic acid (- 5.00 kcal/mole) in their decreasing order. Further, active site residues were identified in both the isoforms and in silico mutation and docking analysis was performed. Our analysis suggested that ASP228, TYR262, and PRO326 for Ll4CL1 and SER165, LYS247 and PRO315 for Ll4CL2 were important for their functional activity. Based on differential substrate preferences of the two isoforms, as a first step towards genetically modified Leuaena having the desired phenotype, it can be proposed that over-expression of Ll4CL1 gene and/or down-regulation of Ll4CL2 gene could yield higher S/G ratio leading to better extractability of lignin.