Abstract
Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SSoNDelta. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SSoNDeltalong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SSoNDelta. Furthermore, SSoNDeltalong and SSoNDelta displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SSoNDeltalong under specific metabolic conditions could be hypothesized.
Highlights
Most esterases/lipases belonging to the hormone-sensitive lipase (HSL) family, comprising either characterized enzymes or open reading frames (ORFs), display similar primary sequences of about 300 amino acids
We performed a comparative analysis with a truncated version of another protein of the HSL family, namely, Alicyclobacillus acidocaldarius esterase 2 (EST2; Manco et al 1997, 1998), and demonstrated that the N-terminus of EST2 is involved in substrate specificity, catalytic efficiency, thermostability, thermophilicity and even regioselectivity (Mandrich et al 2005)
Kim et al (2004) neither purified nor characterized their longer version, they showed that its N-terminus shares weak similarity to other HSL family proteins, and demonstrated by in situ colony assay that the protein has enzymatic activity
Summary
Most esterases/lipases belonging to the hormone-sensitive lipase (HSL) family, comprising either characterized enzymes or open reading frames (ORFs), display similar primary sequences of about 300 amino acids (see the ESTHER database at http://bioweb.ensam.inra.fr/esther). We cloned and characterized an esterase belonging to the HSL family from Sulfolobus solfataricus P2 (She et al 2001), which we named SsoNΔ. Kim et al (2004) reported on the characterization of an esterase, called Est, resulting from the cloning of SSO2493, but they erroneously attributed the sequence of SSO2517 to Est, corresponding to SsoNΔ protein, and the protein sequence of SSO2493 was named Est. Kim et al (2004) successfully cloned SSO2517 from S. solfataricus P2 but with an extra 60 amino acids at the N-terminus compared with the SsoNΔ version that we previously characterized (Mandrich et al 2005). Kim et al (2004) neither purified nor characterized their longer version, they showed that its N-terminus shares weak similarity to other HSL family proteins, and demonstrated by in situ colony assay that the protein has enzymatic activity.
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