Batra-Safferling R

References (4)

Title : Structural, mechanistic and physiological insights into phospholipase A-mediated membrane phospholipid degradation in Pseudomonas aeruginosa - Bleffert_2022_Elife_11_e72824
Author(s) : Bleffert F , Granzin J , Caliskan M , Schott-Verdugo SN , Siebers M , Thiele B , Rahme LG , Felgner S , Dormann P , Gohlke H , Batra-Safferling R , Erich-Jager K , Kovacic F
Ref : Elife , 11 : , 2022
Abstract : Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A(1) (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa deltaplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-A-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.
ESTHER : Bleffert_2022_Elife_11_e72824
PubMedSearch : Bleffert_2022_Elife_11_e72824
PubMedID: 35536643
Gene_locus related to this paper: pseae-PA2949

Title : Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling - Bleffert_2021_Biorxiv__
Author(s) : Bleffert F , Granzin J , Caliskan M , Schott-Verdugo SN , Siebers M , Thiele B , Rahme L , Felgner S , Dormann P , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Biorxiv , : , 2021
Abstract : Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A 1 (PlaF) from Pseudomonas aeruginosa involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa delta plaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-A-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. A dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network which likely explains the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the GPL remodeling pathway for virulence and pave the way for the development of a novel therapeutic class of antibiotics targeting PlaF-mediated membrane GPL remodeling. Synopsis Membrane homeostasis can be regulated by phospholipase-controlled deacylation of endogenous glycerophospholipids (GPLs) followed by reacylation of products, known as the Lands cycle in eukaryotes. Here we show that the human pathogen Pseudomonas aeruginosa uses intracellular phospholipase A 1 (PlaF) to modulate membrane GPL composition, which is the first example in bacteria. This newly identified PLA 1 indirectly regulates the bacterial virulence properties by hydrolyzing a specific set of membrane GPLs. The crystal structure of full-length PlaF dimers bound to natural ligands, MD simulations, and biochemical approaches provide insights into the molecular mechanism of dimerization-mediated inactivation of this single-pass transmembrane PLA 1 . Our findings shed light on a mechanism by which bacterial intracellular PLAs might regulate membrane homeostasis what showcases these enzymes as a promising target for a new class of antibiotics.
ESTHER : Bleffert_2021_Biorxiv__
PubMedSearch : Bleffert_2021_Biorxiv__
PubMedID:
Gene_locus related to this paper: pseae-PA2949

Title : Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization - Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
Author(s) : Bleffert F , Granzin J , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Acta Crystallographica F Struct Biol Commun , 75 :270 , 2019
Abstract : The human membrane-bound alpha/beta-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 beta-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 A. 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.
ESTHER : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedSearch : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedID: 30950828
Gene_locus related to this paper: pseae-PA2949

Title : A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli - Kovacic_2016_FEBS.Open.Bio_6_484
Author(s) : Kovacic F , Bleffert F , Caliskan M , Wilhelm S , Granzin J , Batra-Safferling R , Jaeger KE
Ref : FEBS Open Bio , 6 :484 , 2016
Abstract : Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of beta-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.
ESTHER : Kovacic_2016_FEBS.Open.Bio_6_484
PubMedSearch : Kovacic_2016_FEBS.Open.Bio_6_484
PubMedID: 27419054
Gene_locus related to this paper: pseae-PA2949