Characterization of a novel thiol peroxidase (BsYmaD) from Bacillus subtilis

• Grant number: 24/14130-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2024
• End date: August 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Laura Maria Batista Leal
• 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

The Ohr / OsmC is a super-family of proteins consists of three sub-groups: Ohr; OsmC and Ohr-like. Ohr ("Organic Hydroperoxide Resistance proteins") are thiol-dependent peroxidases that efficiently reduce organic hydroperoxides, but not H2O2. These proteins are primarily found in bacteria and also in other microbial eukaryotes, while absent in vertebrates and vascular plants, making them promising targets for drug development. ”ohr strains are typically resistant to H2O2, while sensitive to organic hydroperoxides. However, a null mutant for an Ohr-like protein (YmaD) in Bacillus subtilis displayed high sensitivity to H2O2, while it was resistant to organic hydroperoxides. This finding prompted us to investigate the substrate specificities of recombinant YmaD, which was expressed in Escherichia Coli and purified by nickel affinity chromatography, using a Akta FPLC system. Then, enzymatic activity of YmaD was analyzed by the lipoamide - lipoamide dehydrogenase assay. Previous results by our group indicated that BsYmaD is one to two orders of magnitude less reactive (kcat/KM) than Ohrs from B. subtilis (BsOhrA and BsOhrB), following a Michaelis-Menten approach. In addition, YmaD displayed higher catalytic efficiently towards organic peroxides (tert-butyl and cumene hydroperoxide) than towards H2O2. Next, we intend to characterize the enzymatic activities of YmaD supported by the lipoylated systems of B. subtilis. The investigation on YmaD may lead to a better understanding on the mechanisms by which B. subtilis, a model gram positive bacteria, respond to oxidative stress.