Structure of EstA esterase from psychrotrophic<i>Pseudoalteromonas</i>sp. 643A covalently inhibited by monoethylphosphonate

Brzuszkiewicz, Anna; Nowak, Elżbieta; Dauter, Zbigniew; Dauter, Mirosława; Cieśliński, Hubert; Długołęcka, Anna; Kur, Józef

doi:10.1107/s1744309109030826

Cited by 19 publications

(21 citation statements)

References 25 publications

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“…The overall fold of TesA is similar to previously determined structures of enzymes belonging to the GDSL lipase family with a characteristic α/β/α-fold [6,9–11]. The compact single domain structure is comprised of a central five-stranded β-sheet surrounded by five α-helices and three 3 10 helices.…”

Section: Resultssupporting

confidence: 73%

“…In general, TesA shows low sequence homology to other GDSL hydrolases (7–27% identity) with the exceptions of TAP from E. coli [7] and EstA [9] from the evolutionarily related bacterium Pseudoalteromonas sp. 643A which show 49% and 42% sequence identity, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…This conclusion was based on the crystal structures of TAP complexes where the loop movement takes place in the presence of OCA, but not with bound inhibitor, diethyl p -nitrophenyl phosphate (DEP) with an acyl chain length of 2 carbon atoms [7]. In contrast, in the previously reported structure of Pseudoalteromonas EstA bound to the inhibitor monoethyl p -nitrophenyl phosphate [9], the conformation of the switch loop is similar to TAP-OCA (long acyl chain length) and not to TAP apo state or TAP-DEP (short acyl chain length).…”

Section: Resultsmentioning

confidence: 99%

“…To date, only few structural studies of GDSL hydrolases have been reported [6,9–11]. Despite less than 20% sequence identity, their overall fold proved to be conserved.…”

Section: Introductionmentioning

confidence: 99%

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Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

et al. 2013

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TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…To date, only few structural studies of GDSL hydrolases have been reported [6,9–11]. Despite less than 20% sequence identity, their overall fold proved to be conserved.…”

Section: Introductionmentioning

confidence: 99%

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Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

et al. 2013

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“…25 The transfer of the ethyl group of DNPP has only been previously been reported three times to our knowledge, first in the structure of aged tabuninhibited murine acetylcholinesterase (PDB code 2c0p), 30 second in the esterase EstA from the coldadapted bacterium Pseudoalteromonas sp. 643A (PDB code 3hp4) 31 which has a classic lipase fold and third in a deposited but unpublished structure of FsC (PDB code 3qpa). In these structures, the catalytic triad is correctly formed.…”

Section: Structure Of Hicmentioning

confidence: 99%

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

et al. 2014

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The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K a % 10 5 M 21 ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. TheAbbreviations: AA, all-atom; AoC, Aspergillus oryzae cutinase; AOT, sodium bis(2-ethylhexyl) sulfosuccinate; cmc, critical micelle concentration; DEP, diethylphosphate; DNPP, diethyl p-nitrophenyl phosphate; EDTA, ethylene diamine tetraacetate; FsC, Fusarium solani cutinase; GcC, Glomerella cingulata cutinase; HiC, Humicola insolens cutinase; HSQC, heteronuclear singlequantum coherence; IPTG, isopropyl b-D-1-thiogalactopyranoside; ITC, isothermal titration calorimetry; MD, molecular dynamics; MEP, monoethylphosphate; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; RMSD, root mean square deviation; RMSF, root mean square fluctuation; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; SANS, small-angle neutron scattering; SDS, sodium dodecyl sulfate.Additional Supporting Information may be found in the online version of this article.An interactive view is available in the electronic version of the article. enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

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Thioesterase enzyme families: Functions, structures, and mechanisms

et al. 2022

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Thioesterases are enzymes that hydrolyze thioester bonds in numerous biochemical pathways, for example in fatty acid synthesis. This work reports known functions, structures, and mechanisms of updated thioesterase enzyme families, which are classified into 35 families based on sequence similarity.Each thioesterase family is based on at least one experimentally characterized enzyme, and most families have enzymes that have been crystallized and their tertiary structure resolved. Classifying thioesterases into families allows to predict tertiary structures and infer catalytic residues and mechanisms of all sequences in a family, which is particularly useful because the majority of known protein sequence have no experimental characterization. Phylogenetic analysis of experimentally characterized thioesterases that have structures with the two main structural folds reveal convergent and divergent evolution. Based on tertiary structure superimposition, catalytic residues are predicted.

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Structure of EstA esterase from psychrotrophicPseudoalteromonassp. 643A covalently inhibited by monoethylphosphonate

Cited by 19 publications

References 25 publications

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

Thioesterase enzyme families: Functions, structures, and mechanisms

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