Biochemical, cellular and gene expression characterization of Ohr from Physcomitrella patens, a model organism of plants

• Grant number: 22/13032-7
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: November 01, 2022
• End date: August 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Rebeca Bandeira Candia
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Ohr enzymes are thiol-dependent peroxidases, characterized by two highly conserved cysteine residues and efficient organic peroxide detoxification. The first Ohr enzyme was discovered in the phytopathogen Xanthomonas campestris, then followed by the identification of many Ohr proteins in prokaryotes, which were considered the only type of organisms having this enzyme. However, a recent study in our group showed the occurrence of dozens of Ohr homologues in eukaryotes, including a single plant species, Physcomitrella patens, a plant model organism. In this context, we intend to identify, through bioinformatics, the expression pattern of ohr, also studying the gene expression by quantitative PCR and Western blot. We also intend to determine kinetic parameters of Ohr from P. patens (PpOhr), using a recombinant protein already obtained by Rebeca Candia during her undergraduate studies. Lipoamide-lipoamide dehydrogenase coupled and competitive assays will be carried out. The subcelular localization of PpOhr will also be studied through cellular fractionation and Western Blot experiments, by which we can test the previous bioinformatics predictions. We will also attempt to obtain high quality crystals for X-ray diffraction analysis, so we can elucidate the PpOhr structure. Finally, we will clone and express the Ohr-like enzyme from P. patens (PpOhr-like) in E coli to biochemically characterize this protein. With this study, we expect to gain insights on the cellular roles of PpOhr and PpOhr in plants. (AU)