Est50 is partial in AY186197 and contains also Est30 sequence Q8GCC7 from Ewis H.E., Lu C.-D., Abdelal A.T.is complete . The short partial sequence Tok19A1 esterase Trembl Q9R4I7 is 3 aa different . Q8GCC7 from Ewis et al. Comment this esterase is called est55 in Ewis_2004_Gene_329_187 . Other strain: Bacillus stearothermophilus
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Bacilli: NE > Bacillales: NE > Bacillaceae: NE > Geobacillus: NE > Geobacillus stearothermophilus: NE
Warning: This entry is a compilation of different species or line or strain with more than 90% amino acide identity. You can retrieve all strain data
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) Geobacillus stearothermophilus 10: N, E.
Geobacillus stearothermophilus ATCC 12980: N, E.
Geobacillus sp. 15: N, E.
Geobacillus sp. 12AMOR1: N, E.
Geobacillus sp. LC300: N, E.
Geobacillus sp. BCO2: N, E.
Geobacillus sp. Sah69: N, E.
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MERTVVETRYGRLRGEMNEGVFVWKGIPYAKAPVGERRFLPPEPPDAWDG VREATSFGPVVMQPSDPIFSGLLGRMSEAPSEDGLYLNIWSPAADGKKRP VLFWIHGGAFLFGSGSSPWYDGTAFAKHGDVVVVTINYRMNVFGFLHLGD SFGEAYAQAGNLGILDQVAALRWVKENIAAFGGDPDNITIFGESAGAASV GVLLSLPEASGLFRRAMLQSGSGSLLLRSPETAMAMTERILDKAGIRPGD RERLLSIPAEELLRAALSLGPGVMYGPVVDGRVLRRHPIEALRYGAASGI PILIGVTKDEYNLFTLTDPSWTKLGEKELLDRINREVGPVPEEAIRYYKE TAEPSAPTWQTWLRIMTYRVFVEGMLRTADAQAAQGADVYMYRFDYETPV FGGQLKACHALELPFVFHNLHQPGVANFVGNRPEREAIANEMHYAWLSFA RTGDPNGAHLPEAWPAYTNERKAAFVFSAASHVEDDPFGRERAAWQGR
References
Title: Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11 Liu P, Ewis HE, Tai PC, Lu CD, Weber IT Ref: Journal of Molecular Biology, 367:212, 2007 : PubMed
Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.
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
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: Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity Wood AN, Fernandez-Lafuente R, Cowan DA Ref: Enzyme Microb Technol, 17:816, 1995 : PubMed
An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8, and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent. The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).
Geobacillus stearothermophilus (Bacillus stearothermophilus) thermostable carboxylesterase Est55 Est-55 Est50 ATCC 12980 IS4712-like insertion
