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- Title
Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima.
- Authors
Levisson, Mark; van der Oost, John; Kengen, Servé W. M.
- Abstract
A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene ( estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4–C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 °C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 °C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.
- Subjects
ESTERASES; ESCHERICHIA coli; GRAM-negative bacteria; LIPASES; GENES; CLONING
- Publication
FEBS Journal, 2007, Vol 274, Issue 11, p2832
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2007.05817.x