Molecular modeling of GcoA enzyme activity on lignin

• Grant number: 21/10472-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): March 01, 2022
• Effective date (End): February 28, 2023
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Munir Salomao Skaf
• Grantee: Artur Hermano Sampaio Dias
• Supervisor: Samuel de Visser
• Host Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Research place: University of Manchester, England
• Associated to the scholarship: 19/17350-0 - Molecular modeling of carbohydrate-modifying enzymes, BP.DR

Abstract

Lignin is a heterogeneous biopolymer present in plant cell walls and plays a major role in providing structural strength to plants and facilitating transportation of water and nutrients. Given its abundance (the second most abundant biopolymer on Earth), lignin is the major source of aromatic carbon on the planet, which highlights the opportunity of harnessing this renewable source for valuable chemicals. The lignin monomers can be easily converted into high-value compounds - ranging from alternatives to petroleum-based chemicals to pharmaceutical molecules - in many industrial settings. However, currently the vast amounts of lignin generated from agriculture and industry are simply wasted, due to the lack of strategies for its valorization. Thus, investigating and developing approaches for chemical functionalization of lignin monomers is of paramount relevance for creating a greener productive line. Recent studies have elucidated many aspects of an oxidative enzyme from Amycolatopsis sp, named GcoA, which belongs to the class of P450 cytochromes and is able to demethylate lignin constituents to generate catechol, which can be further functionalized for lignin upgrading. The present BEPE proposal aims to support a 12-month PhD student internship with Professor Samuel De Visser at the University of Manchester to carry out studies about GcoA activity on lignin components. Dr. De Visser is a well-known scientist at the Department of Chemical Engineering & Analytical Science and is also affiliated with the internationally renowned Manchester Institute of Biotechnology. Dr. De Visser's Group has come to the spotlight in the area of P450 cytochromes, particularly due to their strong, relevant and pioneering Density Functional Theory (DFT)-based studies with many metalloenzymes, among which is GcoA. We propose to study and design GcoA variants that are more selective towards other lignin small components and derivatives that have not yet been tested, such as guaethol, anisole, and 3-methylcatechol. We will also study the reaction free energy profiles of wild-type GcoA and the proposed mutants using classical and quantum-classical MD techniques, as well as full quantum approaches based on DFT methods. If time allows, we consider engineering GcoA to enlarge its substrate binding pocket, making it able to comport larger portions of lignin. For the engineering of GcoA, initially the substrate binding site will be thoroughly scrutinized to look for hot-spots that could be mutated. This fundamental step will be guided by residue conservation analysis and by the results of previous works that have engineered GcoA. The proposed mutations in the active site will aim at creating polar contacts that stabilize the transition state of a particular lignin constituent - elucidated by the full-quantum calculations, thus increasing the enzyme's selectivity towards a particular monomer. Additionally, advanced techniques that look into the free energy profile of substrate binding will be used to compare free energy profiles between wild-type and mutant GcoA, as well as to search for mutants that better bind to the lignin monomers in question and look for molecular reasons that drive enzyme-substrate association. If time allows it, the enlargement of the enzyme's binding site will be carried out in a similar fashion: starting from the residue conservation analysis, we will look into mutations that may broaden the binding cleft without major loss of activity. Finally, this BEPE proposal is aligned with the global goal of creating an environmentally-friendly economy. Developing technologies to enable reinsertion of waste by-products in the productive line, thus reducing the amount of waste and the extraction rates, is a promising means for developing a more sustainable economy. This project aims at helping to reach this goal through cutting-edge science using state-of-art computational simulations that may elucidate mechanisms for lignin valorization. (AU)