Abstract
P. aeruginosa and N. gonorrhoeae rank high on the priority lists of the World Health Organization (WHO) and the Centres for Disease Control and Prevention (CDC) as important targets for antimicrobial drug discovery due to their high incidence of resistance to antibiotics. N. gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhea, is a gram-negative diplococcus found inside and outside various human cell types. P. aeruginosa, an opportunistic gram-negative bacterium, is a significant human pathogen that commonly infects individuals with compromised immune systems, such as those with eye injuries, HIV-1, cancer, surgical or burn wounds, or cystic fibrosis (CF).
The cell wall of bacteria is composed of a membrane and peptidoglycan (PG) layer. The PG layer is a combination of sugars and amino acids. Targeting crucial enzymes in the initial stages of peptidoglycan biosynthesis is frequently proposed as a promising strategy for developing new inhibitors to combat human infectious diseases. One such enzyme, glutamate racemase (EC 5.1.1.3), commonly referred to as MurI, plays a pivotal role in this phase of peptidoglycan synthesis, being essential to produce D-glutamate (D-Glu), a key component of peptidoglycan. In some bacteria, including P. aeruginosa and N. gonorrhoeae, glutamate racemase (MurI) stands as the sole enzyme responsible for converting L-Glu into D-Glu. Therefore, MurI inhibition may be a promising approach to combat P. aeruginosa and N. gonorrhoeae.
To facilitate research on MurIPA and MurING, both enzymes were produced in a soluble form by overexpressing them in E. coli and then purified using affinity and size exclusion chromatography columns. Optimal buffers for each enzyme were determined through thermal shift assays. The impacts of the D-Glu and related compounds on the enzymes were also investigated using thermal shift assays. Additionally, the racemase activity of both enzymes was evaluated using a coupled enzyme assay.
Moreover, this study yielded five high-resolution crystal structures. Initially, the structure of Apo-MurIPA in complex with D-Glu was successfully determined to a resolution of 1.74 Å. Subsequently, the structure of the MurIPA-UDP-MurNAcA complex was solved to a resolution of 1.73 Å. By comparing the structures with and without the activator bound, we were able to observe conformational changes in the MurIPA structure, providing insights into the activation mechanism.
To identify potential inhibitors, a screening of 133 available compounds was performed on MurIPA, utilizing various methods such as thermal shift assays, and IC50 measurements. Among the tested compounds, some inhibitors displayed inhibition potential, as evidenced by either inducing a shift in the thermal stabilization of MurIPA or exhibiting an IC50 value. Notably, the β-Chloro-D-alanine (BCDA) inhibitor demonstrated the lowest IC50 value of 4 µM. To gain insights into the mechanism of BCDA inhibition, the structure of the Apo-MurIPA-BCDA complex was resolved to a resolution of 2.0 Å. This structure revealed that the BCDA inhibitor binds covalently to Cys186, resulting in the inactivation of the enzyme.
Likewise, the high-resolution crystal structure of MurING was successfully determined to a resolution of 1.95 Å. To gain further insights into the quaternary structure of MurING, various techniques were employed, including dynamic light scattering, negative staining, and small-angle X-ray scattering. However, MurING did not show any enzyme activity.
Overall, the research on MurIPA and MurING has yielded promising results regarding the structure and function of glutamate racemases and their significance as potential drug targets. Ongoing investigations into these enzymes can potentially contribute to the development of innovative therapeutic approaches to combat P. aeruginosa infections and treat gonorrhea caused by N. gonorrhoeae.