PDBsum entry 2ogs

Hydrolase

PDB id: 2ogs

Contents

Protein chain

479 a.a. *

Metals

IOD ×3

Waters ×151

* Residue conservation analysis

PDB id:

2ogs

Name:

Hydrolase

Title:

Crystal structure of the geobacillus stearothermophilus carboxylesterase est55 at ph 6.2

Structure:

Thermostable carboxylesterase est50. Chain: a. Engineered: yes

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.02Å R-factor: 0.204 R-free: 0.236

Authors:

P.Liu,H.E.Ewis,P.C.Tai,C.D.Lu,I.T.Weber

Key ref:

P.Liu et al. (2007). Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11. J Mol Biol, 367, 212-223. PubMed id: 17239398 DOI: 10.1016/j.jmb.2006.12.067

Date:

08-Jan-07 Release date: 13-Feb-07

Protein chain

Q8GCC7  (Q8GCC7_GEOSE) -  Carboxylic ester hydrolase from Geobacillus stearothermophilus

Seq: 498 a.a.

Struc: 479 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.1.- - ?????

DOI no: 10.1016/j.jmb.2006.12.067

J Mol Biol 367:212-223 (2007)

PubMed id: 17239398

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11.

P.Liu, H.E.Ewis, P.C.Tai, C.D.Lu, I.T.Weber.

ABSTRACT

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.

Selected figure(s)

Figure 2.
Figure 2. Superposition of Est55 and human carboxylesterase hCE1 structures. Est55 is in green and hCE1 is in red. The active sites are within the ellipse. The black arrow indicates the regulatory domain, which shows significant differences between the two structures. Figure 2. Superposition of Est55 and human carboxylesterase hCE1 structures. Est55 is in green and hCE1 is in red. The active sites are within the ellipse. The black arrow indicates the regulatory domain, which shows significant differences between the two structures.

Figure 4.
Figure 4. (a) The active site region of Est55. Inter-atomic interactions are shown as broken lines with distances in Å. The catalytic residues Ser194, Glu310 and His409 are labeled in red. (b) Comparison of active sites of Est55 (green) and hCE1 (red). The catalytic triad residues of hCE1 are Ser221, Glu354, and His468. Cys408 in Est55 is equivalent to Asp467 in hCE1. Figure 4. (a) The active site region of Est55. Inter-atomic interactions are shown as broken lines with distances in Å. The catalytic residues Ser194, Glu310 and His409 are labeled in red. (b) Comparison of active sites of Est55 (green) and hCE1 (red). The catalytic triad residues of hCE1 are Ser221, Glu354, and His468. Cys408 in Est55 is equivalent to Asp467 in hCE1.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 367, 212-223) copyright 2007.

Figures were selected by an automated process.