Structure Report for: 1TQH

Liu, P., Wang, Y.F., Ewis, H.E., Abdelal, A.T., Lu, C.D., Harrison, R.W., Weber, I.T.

Title: Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus
Ewis HE, Abdelal AT, Lu CD
Ref: Gene, 329:187, 2004 : PubMed
Abstract


ESTHER: Ewis_2004_Gene_329_187
PubMedSearch: Ewis 2004 Gene 329 187
PubMedID: 15033540
Gene_locus related to this paper: geost-est30, geost-est50

Abstract

Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries.



        

Title: Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30
Liu P, Wang YF, Ewis HE, Abdelal AT, Lu CD, Harrison RW, Weber IT
Ref: Journal of Molecular Biology, 342:551, 2004 : PubMed
Abstract


ESTHER: Liu_2004_J.Mol.Biol_342_551
PubMedSearch: Liu 2004 J.Mol.Biol 342 551
PubMedID: 15327954
Gene_locus related to this paper: geost-est30

Abstract

Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.