composed of an N-terminal carboxylesterase domain and a C-terminal OsmC-like domain (osmotically induced family of proteins-like domain. This domain is not related to ABHD and excluded here. E.coli OsmC protein is able to metabolise peroxide, but in the Est-OsmC this activity is not found). Members of the family contained a second, less well conserved,GxSxG pentapeptide found 32 residues upstream of the completely conserved pentapeptide containing the active Serine. Two cysteine residues included in a putative substrate-binding region are found in the N-terminus.
Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical alpha/beta hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.
A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).
