Crystallization and biophysical characterization of oxidative enzymes active in fatty acids

• Grant number: 21/10324-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2022
• End date: June 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Leticia Maria Zanphorlin
• Grantee: Raul de Castro Cunha Claudino
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

Terminal alkenes (or olefins) constitute an industrially important chemical platform for producing polymers, fertilizers, lubricants, surfactants, and especially drop-in biofuels, as they are molecules functionally similar to those of fossil origin. Currently, alkenes are produced through chemical conversion, that is, cracking under high temperature and pressure conditions, using petroleum-based feedstock. Some recently discovered microorganisms, especially the bacteria of the genus Jeotgalicoccus and Pseudomonas, produce alkenes naturally. Their genomes contain the enzymes CYP152 and UndA, respectively, which can decarboxylate fatty acids, making 1-alkenes, which could act as a biological replacement platform for oil. Rare UndA-type enzymes with decarboxylation activity have been discovered, and only one has elucidated tertiary structure (PDB 4WWJ). Therefore, knowledge about the molecular mechanisms of these decarboxylases is still very scarce, mainly due to the lack of structural information. To discover new decarboxylases, our research group carried out a microbial consortium in the presence of different sources of fatty acids. Interestingly, genomic sequencing revealed that microorganisms of the Proteobacteria phylum have in their genomes orthologous UndA enzymes with 23 and 81% identity in the primary sequence. The genes were acquired commercially and initially tested in our laboratory, being produced heterologously in solution. Therefore, this scientific initiation project aims to optimize the expression and purification protocols of selected decarboxylases to obtain the desired amount for high-resolution structural studies by X-ray crystallography. In addition, perform biophysical studies using circular dichroism and dynamic light scattering techniques in the presence and absence of fatty acids of different sizes. The results may help us understand the relationship between the structure and function of these enzymes and their molecular mechanisms. Finally, this project has enormous potential to contribute to developing biological routes to produce drop-in biofuels and other bioproducts derived from terminal alkenes.(AU)