Structure Report for: 3DOI

Levisson, M., Sun, L., Hendriks, S., Swinkels, P., Akveld, T., Bultema, J.B., Barendregt, A., van den Heuvel, R.H.H., Dijkstra, B.W., van der Oost, J., Kengen, S.W.M

Title: Crystal structure and biochemical properties of a novel thermostable esterase containing an immunoglobulin-like domain
Levisson M, Sun L, Hendriks S, Swinkels P, Akveld T, Bultema JB, Barendregt A, van den Heuvel RH, Dijkstra BW and Kengen SW <1 more author(s)> Levisson M, Sun L, Hendriks S, Swinkels P, Akveld T, Bultema JB, Barendregt A, van den Heuvel RH, Dijkstra BW, Van der Oost J, Kengen SW (- 1)
Ref: Journal of Molecular Biology, 385:949, 2009 : PubMed
Abstract


ESTHER: Levisson_2009_J.Mol.Biol_385_949
PubMedSearch: Levisson 2009 J.Mol.Biol 385 949
PubMedID: 19013466
Gene_locus related to this paper: thema-TM0033

Abstract

Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 degrees C and at pH 8.5, with a preference for esters with short acyl chains (C2-C10). Its 2.6-A-resolution crystal structure revealed a classical alpha/beta hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-A-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA.



        

Title: Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima
Sun L, Levisson M, Hendriks S, Akveld T, Kengen SW, Dijkstra BW, Van der Oost J
Ref: Acta Crystallographica Sect F Struct Biol Cryst Commun, 63:777, 2007 : PubMed
Abstract


ESTHER: Sun_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_777
PubMedSearch: Sun 2007 Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun 63 777
PubMedID: 17768353
Gene_locus related to this paper: thema-TM0033

Abstract

A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 A resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 A. There are two molecules in the asymmetric unit, with a V(M) of 2.9 A3 Da(-1) and 58% solvent content.