Structural insight of two 4-Coumarate CoA ligase ( 4CL ) isoforms in Leucaena suggests targeted genetic manipulations co
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ORIGINAL ARTICLE
Structural insight of two 4‑Coumarate CoA ligase (4CL) isoforms in Leucaena suggests targeted genetic manipulations could lead to better lignin extractability from the pulp Himanshu Shekhar1 · Gaurav Kant1 · Rahul Tripathi1 · Shivesh Sharma1 · Ashutosh Mani1 · N. K. Singh1 · Sameer Srivastava1 Received: 19 March 2020 / Accepted: 31 July 2020 © King Abdulaziz City for Science and Technology 2020
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. Keywords Leucaena leucocephala · 4-Coumarate CoA ligase · High S/G ratio · In silico mutational analysis · Differential substrate preference
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13205-020-02375-2) contains supplementary material, which is available to authorized users.
Etymologically, the word ‘lignin’ finds its origin in the Latin word ‘lignum’ meaning wood. It is a non-sugar aromatic polymer produced by oxidative combinatorial coupling and is found in the secondary cell wall of plants, where it fills
* Sameer Srivastava [email protected]
Ashutosh Mani [email protected]
Himanshu Shekhar [email protected]
N. K. Singh [email protected]
Gaurav Kant [email protected]
1
Rahul Tripathi [email protected]
Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad 211004, India
Shivesh Sharma shiv
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