Biochemical, biophysical and structural study of a Mur enzyme complex from the operon of Bordetella pertussis

• Grant number: 20/01286-9
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: June 01, 2020
• End date: December 31, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Andrea Dessen de Souza e Silva
• Grantee: Karina Tamie Shirakawa
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

The Peptidoglycan (PG) is one of the main components of the bacterial cell wall, being responsible to maintain the form and integrity of the cell. As an essential component for bacterial survival and found only in prokaryotic organisms, the PG biosynthesis has been widely explored for the development of antibiotics, even in the face of the global crisis caused by the antibiotic resistance increase. PG biosynthesis is a dynamic and highly complex process mediated by a group of proteins localized at different parts of the cell. Mur ligases (MurC, MurD, MurE, and MurF) are cytoplasmatic enzymes that, together with MurG (membrane-associated protein) and MraY (membrane protein), are responsible for the synthesis of the PG precursors. These proteins have been extensively studied and characterized individually, and, even though inhibitors have been found for them in vitro, they do not show clear antibacterial intracellular activity. A hypothesis would be that the Mur ligases form a mega-complex which could facilitate the formation of the PG subunit and, somehow, it could mask the active sites of the proteins. Different research groups demonstrated that the cytoplasmatic proteins of the PG biosynthesis can identify each other, and, recently, our research group identified that the Mur ligases from Streptococcus pneumoniae can interact strongly as binary complexes. However, until now, the complex between these proteins have not been reported. In our preliminary essays, we were able to express and purify at least three Mur ligases together with MurG, showing promising results towards the characterization of a Mur enzyme complex, essential for PG biosynthesis, using an innovative technique: the expression with the operon system naturally found in microorganisms. Biochemical and biophysical techniques will be used to analyze the interactions of the proteins in the complex. Furthermore, crystallization essays and/or electron microscopy will be used to characterize this complex structurally. The results of this project will provide more detailed information on the PG biosynthesis, and the interactions between these proteins, assisting the pharmaceutical industry in the development of more efficient drugs. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SHIRAKAWAA, KARINA T.; SALAA, FERNANDA ANGELICA; MIYACHIROC, MAYARA M.; JOBC, VIVIANA; TRINDADEA, DANIEL MARAGNO; DESSENA, ANDREA; MAYER, CHRISTOPH; KARLS, EBERHARD. Architecture and genomic arrangement of the MurE-MurF bacterial cell wall biosynthesis complex. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v. 120, n. 21, p. 10-pg., 2023-05-15. (11/52067-6, 17/12436-9, 18/16346-7, 20/01286-9)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

SHIRAKAWA, Karina Tamie. Architecture, interactions and genomic analyses of Mur ligases. 2024. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.