Biochemical and Structural Analysis of the Bacterial Enzyme Succinyl-Diaminopimelate Desuccinylase (DapE) from
Emma H Kelley1, George Minasov2,3, Katherine Konczak1
1Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 West Sheridan Road, Chicago, Illinois 60660, United States.
Abstract:
There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).
Insights
New antibiotic targets are crucial due to rising resistance. Researchers studied succinyl-diaminopimelate desuccinylase (DapE) from Acinetobacter baumannii, revealing distinct inhibitor responses and enzyme stability compared to other species.
Area of Science:
- Structural Biology
- Enzymology
- Antimicrobial Resistance
Background:
- Antibiotic resistance necessitates novel therapeutic targets.
- Succinyl-diaminopimelate desuccinylase (DapE) is a promising antibacterial target.
- Understanding species-specific DapE inhibition is key for drug development.
Purpose of the Study:
- To determine the structure of Acinetobacter baumannii DapE (AbDapE).
- To investigate AbDapE inhibition by known inhibitors of Haemophilus influenzae DapE (HiDapE).
- To analyze the impact of inhibitors on AbDapE stability and compare it with HiDapE.
Main Methods:
- X-ray crystallography was used to determine the structures of AbDapE.
- Thermal shift assays were employed to assess enzyme stability.
- Enzyme inhibition assays were performed using known HiDapE inhibitors.
Main Results:
- AbDapE exhibits differential inhibition compared to HiDapE; captopril and sulfate inhibit both, but an indoline sulfonamide inhibits only HiDapE.
- Sulfate decreases AbDapE stability, while captopril increases it, contrasting with their stabilizing effect on HiDapE.
- Two crystal structures of AbDapE were determined, revealing a closed conformation with bound succinate and acetate (PDB 7T1Q) or succinate and lactate (PDB 8F8O).
Conclusions:
- AbDapE displays distinct inhibitor profiles and stability characteristics compared to HiDapE.
- Structural insights into AbDapE provide a basis for developing targeted inhibitors.
- Findings highlight the importance of species-specific studies for antibiotic development against ESKAPE pathogens.
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