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.

4-香豆酸：辅酶 A 连接酶 ( 4CL ) 是参与木质素生物合成途径早期步骤的关键酶。假设调节植物中的 S 和 G 单木质醇含量。去除木质素对于造纸工业来说是必不可少的，更高的 S/G 比控制着更好的木质素可提取性和制浆过程的经济性。这种背景促使我们预测的两个异构体的三维结构4CL在银合欢和评估他们的底物偏好。Ll4CL1和Ll4CL2蛋白的 3D 结构是通过同源建模创建的，并通过环细化进一步细化。分子对接研究表明两种同工型的底物偏好不同。Ll4CL1优选芥子酸(- 4.91 kcal/mole)、阿魏酸(- 4.84 kcal/mole)、羟基阿魏酸(- 4.72 kcal/mole)和咖啡酸(- 4.71 kcal/mole)，按照它们的降序排列。类似地，Ll4CL2优选咖啡酸（- 6.56 kcal/mole，4 H 键）、羟基阿魏酸（- 6.56 kcal/mole，3 H 键）和阿魏酸（- 6.32 kcal/mole）和芥酸（- 5.00 kcal/mole）摩尔）按降序排列。此外，在同种型和计算机突变和对接分析中都鉴定了活性位点残基。我们的分析表明 Ll4CL1 的 ASP228、TYR262 和PRO326和 SER165、Ll4CL2 的 LYS247 和PRO315对它们的功能活动很重要。基于两种同种型的不同底物偏好，作为具有所需表型的转基因银合欢的第一步，可以提出Ll4CL1基因的过表达和/或Ll4CL2基因的下调可以产生更高的 S/G 比从而提高木质素的可提取性。