Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

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.

Novel intracellular phospholipase B fromPseudomonas aeruginosawith activity towards endogenous phospholipids affects biofilm assembly

“…Extracellular PLA/Bs are toxins involved in host cell membrane disruption [62] or modulation of host cell pathways through the release of bioactive compounds [7]. On the other hand, the function of intracellular PLA/Bs in bacteria is still not clearly established although we recently discovered novel cytoplasmic membrane-bound PLA1 PlaF from P. aeruginosa which remodeling of membrane GPLs is suggested as a virulence mechanism [9, 10]. Interestingly, the function of intracellular PLAs for the regulation of fatty acyl chain composition in GPLs through a deacylation-reacylation pathway called Lands' cycle was described in yeast [63] and other eukaryotes [64].…”

“…In conclusion, the ability of PaPlaB to rapidly degrade endogenous GPLs and above-proposed membrane localization of this novel PLB of P. aeruginosa , suggesting that PaPlaB might modulate the molecular GPL profile of P. aeruginosa membranes similarly as described for PlaF [9, 10], PLA 2 from rat [73], yeast [63] and other eukaryotes [64]. This adaptive GPL modulation might indirectly affect biofilm formation of P. aeruginosa what goes along with findings that P. aeruginosa undergoes drastic changes in membrane GPL composition upon transition from the planktonic to a biofilm lifestyle [72].…”

“…The contribution of bacterial phospholipases to virulence is predominantly related to damaging the host cells, which mostly enhances the survival and spread of the pathogen in the host [6, 7]. Several phospholipases of P. aeruginosa , namely phospholipases A ExoU [8] and PlaF [9, 10], phospholipase A/esterase EstA [11], phospholipase C PlcH [12], and two phospholipases D, PldA and PldB [13], were suggested to be virulence factors in that way. The ExoU, PldA, and PldB are directly secreted into eukaryotic cells, where they modulate native host pathways to facilitate invasion by P. aeruginosa or inflammation [14].…”

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

“…The presence of desired nucleotide substitutions was confirmed by DNA sequencing (MWG Biotech, Ebersberg, Germany). PlaF was purified from P. aeruginosa p-plaF membranes and solubilized with DDM, as described previously [109]. Proteins were analyzed by polyacrylamide gel electrophoresis under denaturation conditions (SDS-PAGE) on 14%…”

“…The esterase activities of PlaF and variants were determined with p-NPB as substrate as described previously [111], using a 96-well microplate and starting the reaction by adding 100 µl of PlaF sample (16 nM) to the 100 µl of p-NPB solution (2 mM). Kinetic parameters (K m and k cat ) for hydrolysis of p-NPB were determined using 8 nM enzyme as described previously [109]. Kinetic parameters were determined by non-linear regression analysis of data fitted to the Michaelis-Menten equation with PrismLab.…”