Expression, purification and crystallization of acetyl-CoA hydrolase fromNeisseria meningitidis

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.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase

“…The expression, purification and crystallization of NmACT-WT, NmACT-Cys 158 -X, NmACT-N24A, NmACT-D39A, and NmACT-Cys 158 -X:R93E were carried out as described in our crystallization report (7). All constructs were co-crystallized with CoA in multiple crystallizing conditions as reported for NmACT-WT (7).…”

“…The expression, purification and crystallization of NmACT-WT, NmACT-Cys 158 -X, NmACT-N24A, NmACT-D39A, and NmACT-Cys 158 -X:R93E were carried out as described in our crystallization report (7). All constructs were co-crystallized with CoA in multiple crystallizing conditions as reported for NmACT-WT (7). Crystals grown in 100 mM Tris, pH 8.5, and 2 M ammonium phosphate led to complete datasets for NmACT-WT, NmACT-Cys 158 -X, NmACT-N24A, NmACT-D39A, and NmACT-Cys 158 -X:R93E, and diffraction to 2.0, 2.3, 2.0, 2.8, and 2.8Å, respectively.…”

Drought induces soil microbial stress responses and emissions of volatile organic compounds in an artificial tropical rainforest

“…ACH1 was also positively correlated with acetate-C2 e ux (ρ = 0.59; p < 0.01) (Table 1), perhaps due to its direct role in acetate production, or consumption if increased acetate concentrations stimulate its activity. While genes encoding for other acetyl-CoA hydrolases are present in bacteria 32 , ACH1 is only present in the mitochondria 33 of eukaryotes, including fungi 34,35 . ACH1 encodes for a gene with sequence similarity to either an acetyl-CoA hydrolase, which can produce acetate and CoA 35,36 and may play a role in transporting acetate from acetyl-CoA across organelle membrane to cytosol for conversion back to acetyl-CoA 35,36 by acetyl-CoA synthetase (acs; K01895), or a a CoA-transferase which can transfer CoA from succinyl-CoA to acetate, thus consuming acetate and playing a role in acetate detoxi cation 34 .…”