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Updated: May 6, 2026
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Expression, purification and crystallization of acetyl-CoA hydrolase from Neisseria meningitidis
Yogesh B Khandokar1, Avinash Londhe, Shilpa Patil
1School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2650, Australia.
Abstract:
Neisseria meningitidis is the causative microorganism of many human diseases, including bacterial meningitis; together with Streptococcus pneumoniae, it accounts for approximately 80% of bacterial meningitis infections. The emergence of antibiotic-resistant strains of N. meningitidis has created a strong urgency for the development of new therapeutics, and the high-resolution structural elucidation of enzymes involved in cell metabolism represents a platform for drug development. Acetyl-CoA hydrolase is involved in multiple functions in the bacterial cell, including membrane synthesis, fatty-acid and lipid metabolism, gene regulation and signal transduction. Here, the first recombinant protein expression, purification and crystallization of a hexameric acetyl-CoA hydrolase from N. meningitidis are reported. This protein was crystallized using the hanging-drop vapour-diffusion technique at pH 8.5 and 290 K using ammonium phosphate as a precipitant. Optimized crystals diffracted to 2.0 Å resolution at the Australian Synchrotron and belonged to space group P2(1)3 (unit-cell parameters a = b = c = 152.2 Å), with four molecules in the asymmetric unit.
Insights
Researchers report the first recombinant expression and crystallization of hexameric acetyl-CoA hydrolase from Neisseria meningitidis. This structural study provides a foundation for developing new therapeutics against antibiotic-resistant bacterial meningitis strains.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Neisseria meningitidis causes bacterial meningitis, with increasing antibiotic resistance necessitating new drug targets.
- Acetyl-CoA hydrolase is crucial for bacterial cell functions, including metabolism and gene regulation.
- Structural elucidation of bacterial enzymes aids in developing novel therapeutics.
Purpose of the Study:
- To report the first recombinant protein expression, purification, and crystallization of hexameric acetyl-CoA hydrolase from Neisseria meningitidis.
- To provide a structural basis for understanding the enzyme's function and potential as a drug target.
Main Methods:
- Recombinant protein expression and purification of N. meningitidis acetyl-CoA hydrolase.
- Crystallization using the hanging-drop vapour-diffusion technique at pH 8.5 and 290 K.
- X-ray diffraction data collection at the Australian Synchrotron to 2.0 Å resolution.
Main Results:
- Successful expression, purification, and crystallization of hexameric acetyl-CoA hydrolase.
- Crystals belonged to space group P2(1)3 with unit-cell parameters a = b = c = 152.2 Å.
- The structure revealed four molecules in the asymmetric unit, diffracting to 2.0 Å resolution.
Conclusions:
- The study presents the first high-resolution crystal structure of N. meningitidis acetyl-CoA hydrolase.
- This structural information is vital for structure-based drug design against N. meningitidis.
- The findings contribute to the development of new strategies to combat antibiotic-resistant meningitis.
