PhD Thesis Defence Presentations - Christos Kosinas

Presentation Title (Τίτλος Παρουσίασης): Mechanistic insights into lignocellulose-degrading enzymes using structural biology and protein engineering methods
Presentation Type (Τύπος Παρουσίασης): PhD Thesis Defence Presentations
Speakers Full Name (Ονοματεπώνυμο): Christos Kosinas
Speakers Affiliation (Προέλευση Ομιλητή): Πανεπιστήμιο Πατρών, Τμήμα Χημικών Μηχανικών
Seminar Room (Αίθουσα): "A. C. Payatakes" Library
Event Date: Wed, Jan 29 2025, Time: 12:00 - 14:30
Live webConference Link: https://upatras-gr.zoom.us/j/92367451107?pwd=Vo1hk1nK7gy8iLgTa2fxqlALuVGnVz.1
Abstract (Περίληψη)

Enzymatic degradation of lignocellulosic biomass has gained increasing interest in recent years, complementing existing chemical and mechanical pretreatment processes. Investigating the mechanism of enzyme activity and designing improved biocatalysts through protein engineering methods can enhance their effective application in industrial processes.

In this doctoral dissertation, four enzymes derived from the thermophilic fungus Thermothelomyces thermophilus were examined. These enzymes contribute to the degradation of the three main components of lignocellulose: cellulose, hemicellulose, and lignin. The study of these specific biocatalysts was conducted by correlating their structure and function properties. Structural studies primarily utilized X-ray crystallography, along with molecular biology techniques and computational tools for simulating enzyme-substrate interactions. Specifically, a lytic polysaccharide monooxygenase (TtLPMO9F), which oxidatively cleaves glycosidic bonds in cellulose, was analyzed. By determining its crystallographic structure and conducting biochemical studies on various point variants, the contribution of specific amino acids to catalytic activity was identified. Additionally, the synergistic action of TtLPMO9F with commercial lignocellulose-degrading enzyme formulations was evaluated. Furthermore, the structure of a laccase (TtLMCO1) with notable biotechnological interest was presented, as it can oxidize various phenolic compounds. Docking simulations of the enzyme's interaction with different substrates revealed that the architecture of its substrate-binding region correlates with its preference for ortho-substituted phenolic compounds.

The second part of the dissertation focuses on two hydrolytic enzymes associated with hemicellulose degradation. Specifically, the crystallographic structures of complexes of a xylanase from the GH30_7 subfamily (TtXyn30A) and its inactive mutants with various xylo-oligosaccharide substrates were determined. This enabled the identification of amino acids responsible for the enzyme's dual mode of action. Finally, the first crystal structure of an acetate esterase from the CE16 family (TtCE16B) was resolved. The crystallographic structure of TtCE16B and an inactive mutant (S19A) in complex with an acetate molecule identified the catalytic amino acids forming the active site and the structural features determining the enzyme's preference for soluble xylo-oligosaccharides.

The findings of this study contribute to a deeper understanding of the mechanisms of these specific biocatalysts for the utilization of plant biomass. At the same time, by modifying the enzyme’s architecture using protein engineering methods, new pathways can be opened for their optimal use in industrial processes and new biocatalytic applications.

Speakers Short CV (Σύντομο Βιογραφικό Ομιλητή)

Education:

  • PhD candidate, Department of Chemical Engineering, University of Patras (2020-today)
  • Post-graduate studies in Biological Technology, Department of Biology, University of Patras (2018-2020)
  • Degree in Biology, Department of Biology, University of Patras (2012-2018)

Publications:

  • Kosinas C., Chorozian K., Sandgren M., Topakas E., Dimarogona M. (2024) Mutational study of a lytic polysaccharide monooxygenase from Myceliophthora thermophila (MtLPMO9F): Structural insights into substrate specificity and regioselectivity. International Journal of Biological Macromolecules. 9, 288:138574. 
  • Pentari C., Zerva A., Kosinas C., Karampa P., Puchart V., Dimarogona M., Topakas E. (2024). The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase. Carbohydrate Polymers. 327, 121667.
  • Pentari C., Kosinas C., Nikolaivits E., Dimarogona M., Topakas E. (2024). Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan. Biotechnology and Bioengineering. 121(7):2067-2078.
  • Kosinas C., Zerva A., Topakas E., Dimarogona M. (2023).  Structure–function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role. Acta Crystallographica Section D Structural Biology, 79(7), 641–654. 
  • Nikolaivits E., Pentari C., Kosinas C., Feiler C. G., Spiliopoulou M., Weiss M. S., Dimarogona M., Topakas E. (2021). Unique features of the bifunctional GH30 from Thermothelomyces thermophila revealed by structural and mutational studies. Carbohydrate Polymers.  273, 118553.
  • Ferousi C., Kosinas C., Nikolaivits E., Topakas E. & Dimarogona M. (2023). Crystal structure of the Fusarium oxysporum tannase-like feruloyl esterase FaeC in complex with p-coumaric acid provides insight into ligand binding. FEBS Letters. 597(10):1415-1427.

 

Conference presentations:

  • Kosinas C., Chorozian K., Katsimantos S., Karnaouri A., Sandgren. M., Topakas E.,      Dimarogona M., “Expanding the catalytic potential of a lytic polysaccharide monooxygenase from Thermothelomyces thermophilus: A comprehensive study of point mutations and structural implications” (oral)  14th Panhellenic Scientific Conference in Chemical Engineering, 29-31 May 2024, Thessaloniki, Greece.
  • Kosinas C., Zerva A., Chalima A., Karnaouri A., Topakas E., Sandgren. M., Dimarogona M., “Catalytic potential of fungal oxidative enzymes towards plant biomass degradation” (oral) 11th Hellenic Crystallographic Association (HeCrA), 20-22 October 2023, Larissa, Greece.
  • Pentari C., Zerva A., Kosinas C., Karamba P., Dimarogona M., Puchart V., Topakas E., “The biotechnological potential of a novel CE16 exo-deacetylase from Thermothelomyces thermophilus” (poster), 16th BIOTRANS, 25-29 June, La Rochelle, France.
  • Kosinas C., Nestor G., Chalima A., Karnaouri A., Topakas E., Sandgren. M., Dimarogona M., “Exploration of the molecular determinants of LPMO function through mutagenesis studies of a Thermothelomyces thermophilus AA9” (poster), 3rd LPMO Symposium,  9-11 November 2022, Oslo, Norway.
  • Pentari C., Zerva A., Kosinas C., Dimarogona M., Topakas E., “A novel CE16 acetyl esterase from Thermothelomyces thermophilus” (poster), FEMS Conference on Microbiology, 30 June – 2 July 2022 Belgrad, Serbia.
  • Kosinas C., Zerva A., Karampa P.*, Topakas E., Dimarogona M., “Structural characterization of a novel laccase-like multicopper oxidase from Thermothelomyces thermophilus” (poster), 13rd Panhellenic Scientific Conference in Chemical Engineering, 2-4 June 2022, Patras, Greece.
  • Kosinas C., Pentari C., Zerva A., Topakas E., Dimarogona M. “Combining challenging crystallographic methods for the structural determination of esterase TtCE16 from Thermothelomyces thermophila” (poster) 10th International Conference of the Hellenic Crystallographic Association (HeCrA), 15-17 October 2021, Athens, Greece.
  • Nikolaivits E., Kosinas C., Pentari C., Feiler C., Weiss M.S., Topakas E. & Dimarogona M. “Structural studies of a GH30 xylanase provide insights into its mechanism of action as a potential biocatalyst”, (poster) PDB50: A special symposium celebrating the 50th anniversary of the Protein Data Bank, May 2021, Virtual Symposium.