Architecture, interactions and genomic analyses of Mur ligases

Author(s)

Karina Tamie Shirakawa

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2024-01-31

Examining board members

Andréa Dessen; Priscila Oliveira de Giuseppe; Marcio Vinicius Bertacine Dias; Ana Carolina de Mattos Zeri; Deborah Schechtman

• Advisor: Andréa Dessen

Abstract

The peptidoglycan (PG) is an essential component of the bacterial cell wall, and thus has been one of the main targets for the development of antibiotics since the implementation of penicillin. Mur ligases (MurC, MurD, MurE and MurF) are responsible for the initial steps of PG biosynthesis in the cytoplasm, and due to their similar characteristics and their involvement in a possible cytoplasmic subcomplex, they have been extensively studied as targets for the development of new antibacterial agents. In most bacteria, Mur ligases are expressed in isolated form, as in the case of Streptococcus pneumoniae; however, in some species these enzymes are fused as is the case of murEmurF fusion in Bordetella pertussis. This work aimed to analyze the interactions and isolate a Mur complex from two human pathogens, S. pneumoniae (Gram-positive) and B. pertussis (Gram-negative). We demonstrated the transient interaction between Mur ligases from both S. pneumoniae and B. pertussis using different techniques, presenting the first evidence of this interaction in B. pertussis. Bioinformatics analyses revealed the existence of different Mur fusions, with MurE-MurF being the most abundant one found. These Mur fusions are predicted to have two architectures: with a linker connecting the proteins, or with the proteins juxtaposed connected by their C and N-terminal ends. In this thesis, we describe the first structure of a MurE-MurF fusion from B. pertussis, which displays a particular structure that was not predicted by the databases, representing a sub-architecture of juxtaposed Mur fusions. Furthermore, we identified key residues involved in the interaction between MurE and MurF in MurE-MurF, which display conservation in other MurE-MurF fusions and in the isolated proteins. These results provide important advances in the understanding of the interaction between Mur ligases and in the formation of the cytoplasmatic subcomplex of the PG, which can assist in the studies and development of inhibitors targeting these proteins (AU)

• FAPESP's process: 20/01286-9 - Biochemical, biophysical and structural study of a Mur enzyme complex from the operon of Bordetella pertussis
• Grantee: Karina Tamie Shirakawa
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)