Assessment of the impact of reducing agent types on electron transfer and enzymatic activity of lytic polysaccharide monooxygenases (LPMOs) from Thermothelomyces thermophilus

• Grant number: 24/16094-9
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: March 01, 2025
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Fernando Segato
• Grantee: Martha Inés Vélez Mercado
• Supervisor: Aniko Varnai
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Institution abroad: Norwegian University Of Life Sciences, Norway
• Associated to the scholarship: 22/04713-0 - Exploring the interaction between lytic polysaccharide monooxygenases and its redox partner cellobiose dehydrogenases, BP.DD

Abstract

Since the discovery of oxidative depolymerization of crystalline cellulose by enzymes known as lytic polysaccharide monooxygenases (LPMOs), these enzymes have been integrated into commercial enzyme cocktails, significantly improving overall hydrolytic performance. LPMOs require a polysaccharide substrate and an enzymatic (such as cellobiose dehydrogenase; CDH) or non-enzymatic electron donor. Despite numerous studies on the mode of action of LPMOs, their interactions with the substrate and electron donors, their exact reaction mechanism and variations between LPMOs remain partly unknown. Preliminary analyses using LPMOs and a CDH from Thermothelomyces thermophilus (earlier called Myceliophthora thermophila) as well as other reductants indicated that some LPMOs exhibited activity only with specific reductants. In this research proposal (BEPE), we aim to investigate the effects of various electron donors, including CDH, on their ability to reduce the active-site copper and to drive the action of five recombinantly expressed LPMOs (four AA9 and one AA16 LPMOs) from T. thermophilus using stopped-flow fluorimetry, isothermal titration calorimetry (ITC), high performance anion exchange chromatography (HPAEC-PAD) and mass spectrometry (MALDI-ToF MS). Identifying differences in behavior among these LPMOs will provide insights into the impact of electron donors and interactions with CDH on LPMO functionality. This work could reveal patterns in LPMO-CDH interactions and aid in engineering LPMOs for more efficient electron transfer.