Abstract
The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl β-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.
Highlights
Enzymes of the / -hydrolase superfamily are found in virtually all organisms and have functional implications that are important to human health (Lord et al, 2013) and bacterial pathogenesis (Flores-Dıaz et al, 2016)
The hydrolytic reactions that are catalyzed by / -hydrolases rely on a hydrogen-bond network between the catalytic triad residues that is essential for formation of the nucleophilic serine (Rauwerdink & Kazlauskas, 2015)
P. aeruginosa PA01 cells harboring pBBR-pa2949 (Kovacic, Bleffert et al, 2016) were cultivated overnight in Luria– Bertani (LB) medium supplemented with tetracycline (100 mg mlÀ1) at 37C (Table 1)
Summary
Enzymes of the / -hydrolase superfamily are found in virtually all organisms and have functional implications that are important to human health (Lord et al, 2013) and bacterial pathogenesis (Flores-Dıaz et al, 2016). The canonical / -hydrolase fold consists of up to 11 -strands folded into a central hydrophobic sheet surrounded by flanking -helices (Heikinheimo et al, 1999) This fold provides a scaffold for a structurally conserved active site comprising the catalytic triad (Ser, His, Asp) and two oxyanion-hole residues (Heikinheimo et al, 1999). Mammals express at least 19 / -hydrolases [called / hydrolase domain (ABHD) proteins in the literature], and the biochemical and physiological functions of most of them are largely unknown (Lord et al, 2013). PA2949 is a 34.8 kDa protein that shows esterase activity and is anchored to the E. coli membrane by a putative N-terminal transmembrane domain (Kovacic, Bleffert et al, 2016). The structure of the putatively single-pass integral membrane protein PA2949 should contribute to the understanding of the function of these proteins, with potential therapeutic implications
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