Introduction:

The carboxyvinyl-transferase MurA catalyses the first committed step of the peptidoglycan biosynthesis allowing bacteria to sustain a functional cell wall, thus, it is generally essential. MurA is of medical importance, since it is the molecular target for the broad-spectrum antibiotic fosfomycin (FOS). FOS binds to an active‑site cysteine of MurA, forming a highly stable thioether bond, which covalently blocks the enzyme, preventing substrate binding and keeping MurA in an inactivated state. While Gram‑negative have one isoform, Gram‑positive usually have two or more isoforms of the enzyme. Bacillus subtilis has two MurA isoforms: while MurAA is essential, MurAB is expandable. The reason for B. subtilis having two isoforms of MurA is unknown and their physiological roles, and kinetic and mechanistic differences have not been investigated, so far.

Goal:

Our aim is to gain a better understanding of the MurA isoforms of B. subtilis, in terms of catalytic properties, susceptibility to FOS, and physiological functions.

Materials and methods:

MurAA and MurAB of B. subtilis were cloned, heterologously expressed, purified and biochemically characterized. A malachite‑green based phosphate assay was used to determine their kinetic parameters and the IC50 for FOS. With native mass spectrometry (nMS) we investigated the occupation of the active site cysteine, after incubation with substrate or antibiotic at varying concentrations.

Results:

MurAA appears to have a higher affinity towards phosphoenolpyruvate (PEP) and FOS, while MurAB has a higher maximal velocity. Most interestingly, purified MurAA and MurAB both carried a PEP molecule covalently bound to the active-site cysteine residue, shown with nMS, and we were able to demonstrate that, in this form, both isozymes are no molecular targets for FOS. Addition of second substrate UDP-N-acetylglucosamine triggers the enzymatic reaction, relieves the active site and, in this form, enabled binding of FOS. Further, the two isozymes have differences in their active‑site occupation, where multiple substrates/products are bound to MurAB, but not to MurAA.

Summary:

Using nMS, we were able to directly show, for the first time, that MurA isozymes are isolated from cells in a FOS-insensitive form with a PEP covalently blocking the active site cysteine. Although the MurA isozymes of B. subtilis have rather similar kinetic properties, differences occur in reaction rate and the affinity for PEP and FOS.