A membrane‐bound esterase PA2949 from <i>Pseudomonas aeruginosa</i> is expressed and purified from <i>Escherichia coli</i>

Kovačić, Filip; Bleffert, Florian; Caliskan, Muttalip; Wilhelm, Susanne; Granzin, Joachim; Batra‐Safferling, Renu; Jaeger, Karl‐Erich

doi:10.1002/2211-5463.12061

Cited by 13 publications

(43 citation statements)

References 62 publications

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“…The pathogenicity of P. aeruginosa is predominantly related to the production of a large spectrum of cell-associated and cellsecreted virulence factors, among which are several /-hydrolases (Van Delden & Iglewski, 1998;Bofill et al, 2010). 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). PA2949 contains a typical /-hydrolase Ser-His-Asp catalytic triad, as shown previously in our mutagenesis studies (Kovacic, Bleffert et al, 2016).…”

Section: Introductionsupporting

confidence: 73%

“…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). PA2949 contains a typical /-hydrolase Ser-His-Asp catalytic triad, as shown previously in our mutagenesis studies (Kovacic, Bleffert et al, 2016).…”

Section: Introductionsupporting

confidence: 60%

“…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 ml À1 ) at 37 C (Table 1). This culture was used to inoculate an expression culture in LB medium supplemented with tetracycline (100 mg ml À1 ) to an initial OD 580 nm of 0.05.…”

Section: Overexpression and Purificationmentioning

confidence: 99%

“…The cells were harvested by centrifugation (15 min at 6750g and 4 C), resuspended in 100 mM Tris-HCl buffer pH 8 and disrupted using a French press. PA2949 was purified from the membrane fraction by immobilized metalaffinity chromatography (IMAC) using Ni-NTA agarose (Qiagen, Hilden, Germany) as described previously (Kovacic, Bleffert et al, 2016), with the modification that the Triton X-100 in the elution buffer was replaced by 30 mM n-octyl -d-glucoside (OG). The PA2949 samples eluted from the Ni-NTA column were transferred into 100 mM Tris-HCl buffer pH 8 supplemented with 30 mM n-octyl -d-glucoside (OG) by gel filtration using a PD-10 column (GE Healthcare, Solingen, Germany) according to the manufacturer's protocol.…”

Section: Overexpression and Purificationmentioning

confidence: 99%

“…We recently characterized the /-hydrolase PA2949 from P. aeruginosa (Kovacic, Bleffert et al, 2016). P. aeruginosa is a versatile pathogen that causes infections in mammals, plants and insects (Mahajan-Miklos et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Introductionsupporting

confidence: 73%

Section: Introductionsupporting

confidence: 60%

Section: Overexpression and Purificationmentioning

confidence: 99%

Section: Overexpression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

Wittgens

Kovačić

Müller

et al. 2016

Appl Microbiol Biotechnol

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The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.

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Abridgement of Microbial Esterases and Their Eminent Industrial Endeavors

Akram,

Fatima,

Shabbir

et al. 2024

Mol Biotechnol

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A membrane‐bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli

Cited by 13 publications

References 62 publications

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

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

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

Abridgement of Microbial Esterases and Their Eminent Industrial Endeavors

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