title: First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui.
abstract: A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far-UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25-0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Müller-Santos
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: de Souza
authors.forename: Fabio de O
authors.initials: Fde O
authors.lastname: Pedrosa
authors.forename: David Alexander
authors.initials: DA
authors.lastname: Mitchell
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.forename: Nadia
authors.initials: N
authors.lastname: Krieger
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004950:Esterases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019614:Haloarcula marismortui / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000494:pharmacology; D017510:Protein Folding / Q000187:drug effects*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology*; D055672:Static Electricity; D013379:Substrate Specificity / Q000187:drug effects; D013499:Surface Properties / Q000187:drug effects; D013696:Temperature; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D012965:Sodium Chloride; D011189:Potassium Chloride; D004950:Esterases