tailieunhanh - Báo cáo khoa học: Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima

A bioinformatic screening of the genome of the hyperthermophilic bacter-ium Thermotoga maritimafor 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, func-tionally expressed in Escherichia coliand purified to homogeneity. | ễFEBS Journal Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima Mark Levisson John van der Oost and Serve W. M. Kengen Laboratory of Microbiology Wageningen University the Netherlands Keywords esterase hyperthermophile lipase thermostable Thermotoga maritima Correspondence M. Levisson Laboratory of Microbiology Wageningen University Hesselink van Suchtelenweg 4 6703 CT Wageningen the Netherlands Fax 31 0317 483829 Tel 31 0317 483748 E-mail Website http Received 16 January 2007 revised 30 March 2007 accepted 2 April 2007 doi 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 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 a b-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 .