PDBsum entry 1k4y

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Hydrolase PDB id
Protein chain
501 a.a. *
Waters ×383
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of rabbit liver carboxylesterase in comple piperidino-piperidine
Structure: Liver carboxylesterase. Chain: a. Engineered: yes
Source: Oryctolagus cuniculus. Rabbit. Organism_taxid: 9986. Organ: liver. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: sf9.
2.50Å     R-factor:   0.229     R-free:   0.294
Authors: S.Bencharit,C.L.Morton,E.L.Howard-Williams,M.K.Danks,P.M.Pot M.R.Redinbo
Key ref:
S.Bencharit et al. (2002). Structural insights into CPT-11 activation by mammalian carboxylesterases. Nat Struct Biol, 9, 337-342. PubMed id: 11967565 DOI: 10.1038/nsb790
09-Oct-01     Release date:   01-May-02    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P12337  (EST1_RABIT) -  Liver carboxylesterase 1
565 a.a.
501 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Carboxylesterase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A carboxylic ester + H2O = an alcohol + a carboxylate
carboxylic ester
+ H(2)O
= alcohol
+ carboxylate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     endoplasmic reticulum   2 terms 
  Biological process     metabolic process   1 term 
  Biochemical function     carboxylic ester hydrolase activity     2 terms  


DOI no: 10.1038/nsb790 Nat Struct Biol 9:337-342 (2002)
PubMed id: 11967565  
Structural insights into CPT-11 activation by mammalian carboxylesterases.
S.Bencharit, C.L.Morton, E.L.Howard-Williams, M.K.Danks, P.M.Potter, M.R.Redinbo.
Mammalian carboxylesterases cleave the anticancer prodrug CPT-11 (Irinotecan) into SN-38, a potent topoisomerase I poison, and 4-piperidino-piperidine (4PP). We present the 2.5 A crystal structure of rabbit liver carboxylesterase (rCE), the most efficient enzyme known to activate CPT-11 in this manner, in complex with the leaving group 4PP. 4PP is observed bound adjacent to a high-mannose Asn-linked glycosylation site on the surface of rCE. This product-binding site is separated from the catalytic gorge by a thin wall of amino acid side chains, suggesting that 4PP may be released through this secondary product exit pore. The crystallographic observation of a leaving group bound on the surface of rCE supports the 'back door' product exit site proposed for the acetylcholinesterases. These results may facilitate the design of improved anticancer drugs or enzymes for use in viral-directed cancer cotherapies.
  Selected figure(s)  
Figure 1.
Figure 1. Crystal structure of rabbit liver carboxylesterase. a, Two-step activation of the anticancer topoisomerase I poison CPT-11 to SN-38 (the active metabolite) and 4-piperidino-piperidine (4PP) by carboxylesterases. 4-piperidino-piperidine-carboxylate spontaneously hydrolyzes to 4PP and CO[2] after step 2. b, Structure-based sequence alignments of rabbit CE (rCE), human CE 1 (hCE1) and human intestinal CE (hiCE) obtained with ClustalW40 and refined using the rCE structure. Conserved residues are in black and nonconserved residues in magenta. Dotted lines indicate missing residues in the rCE structure. N-linked glycosylation sequences, disulfide bonds and putative gate residues are framed in black, and members of the catalytic triad are marked with an asterisk. The catalytic domain is blue; the domain, green; and the regulatory domain, red. c, Structure of rabbit liver carboxylesterase indicating the three domains: catalytic, and regulatory. Coloring as in (b), with catalytic residues in green, N-linked glycosyl groups in cyan and disulfide linkages in orange. d, The active site of rCE (green) superimposed on that of two esterases closely related in structure: triacylglycerol hydrolase (PDB entry 1THG; gold) and cholesterol esterase (2BCE; magenta). The catalytic Glu 353 of rCE is rotated away from the active site relative to orientations observed in other esterases. Glu 353 and His 467 lie adjacent to regions of structural disorder in rCE. e, Stereo view of a composite simulated-annealing omit map (cyan; contoured at 1.0 ) and the final [A]-weighted^34 2F[o] - F[c] map (magenta; contoured at 1.0 ) around the Asn 79 glycosylation site (both maps at 2.5 resolution).
Figure 3.
Figure 3. Structural basis of CPT-11 activation by rCE. a, Stereo view of the gate between the active site (green) and bound 4PP molecule (purple). The regulatory domain (red) is composed of helices 9, 10, 11 and 14, and the gate residues are Leu 252, Ser 254, Ile 387 and Leu 424 (cyan). The residues that mark the beginning and end of the disordered regions of the structure (Phe 354, Lys 371, Glu 459 and His 467) are also labeled. b, A proposed mechanism for the activation of CPT-11 by rCE. CPT-11 (orange) enters from the top of the catalytic gorge and fits well into the active site (catalytic Ser 221 and Glu 353 in green). After cleavage, the alcohol product (SN-38; magenta) leaves via the catalytic gorge, while the acyl product (4PP; purple) moves past the gate residues (cyan) and docks adjacent to the regulatory domain (red) on the surface of the molecule.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2002, 9, 337-342) copyright 2002.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20510380 E.T.Williams, H.Wang, S.A.Wrighton, Y.W.Qian, and E.J.Perkins (2010).
Genomic analysis of the carboxylesterases: identification and classification of novel forms.
  Mol Phylogenet Evol, 57, 23-34.  
20552256 G.Thiagarajan, A.Ray, A.Malugin, and H.Ghandehari (2010).
PAMAM-camptothecin conjugate inhibits proliferation and induces nuclear fragmentation in colorectal carcinoma cells.
  Pharm Res, 27, 2307-2316.  
20161041 R.S.Holmes, L.A.Cox, and J.L.Vandeberg (2009).
A new class of mammalian carboxylesterase CES6.
  Comp Biochem Physiol Part D Genomics Proteomics, 4, 209-217.  
19062296 T.Harada, Y.Nakagawa, R.M.Wadkins, P.M.Potter, and C.E.Wheelock (2009).
Comparison of benzil and trifluoromethyl ketone (TFK)-mediated carboxylesterase inhibition using classical and 3D-quantitative structure-activity relationship analysis.
  Bioorg Med Chem, 17, 149-164.  
18023188 C.E.Wheelock, K.Nishi, A.Ying, P.D.Jones, M.E.Colvin, M.M.Olmstead, and B.D.Hammock (2008).
Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH).
  Bioorg Med Chem, 16, 2114-2130.  
18721110 J.M.Hatfield, M.Wierdl, R.M.Wadkins, and P.M.Potter (2008).
Modifications of human carboxylesterase for improved prodrug activation.
  Expert Opin Drug Metab Toxicol, 4, 1153-1165.  
18219308 J.Rautio, H.Kumpulainen, T.Heimbach, R.Oliyai, D.Oh, T.Järvinen, and J.Savolainen (2008).
Prodrugs: design and clinical applications.
  Nat Rev Drug Discov, 7, 255-270.  
18188187 M.Wierdl, L.Tsurkan, J.L.Hyatt, C.C.Edwards, M.J.Hatfield, C.L.Morton, P.J.Houghton, M.K.Danks, M.R.Redinbo, and P.M.Potter (2008).
An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11.
  Cancer Gene Ther, 15, 183-192.  
18163883 T.M.Streit, A.Borazjani, S.E.Lentz, M.Wierdl, P.M.Potter, S.R.Gwaltney, and M.K.Ross (2008).
Evaluation of the 'side door' in carboxylesterase-mediated catalysis and inhibition.
  Biol Chem, 389, 149-162.  
17264798 K.Tanimoto, M.Kaneyasu, T.Shimokuni, K.Hiyama, and M.Nishiyama (2007).
Human carboxylesterase 1A2 expressed from carboxylesterase 1A1 and 1A2 genes is a potent predictor of CPT-11 cytotoxicity in vitro.
  Pharmacogenet Genomics, 17, 1.  
17239398 P.Liu, H.E.Ewis, P.C.Tai, C.D.Lu, and I.T.Weber (2007).
Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11.
  J Mol Biol, 367, 212-223.
PDB codes: 2ogs 2ogt
17167034 R.M.Wadkins, J.L.Hyatt, C.C.Edwards, L.Tsurkan, M.R.Redinbo, C.E.Wheelock, P.D.Jones, B.D.Hammock, and P.M.Potter (2007).
Analysis of mammalian carboxylesterase inhibition by trifluoromethylketone-containing compounds.
  Mol Pharmacol, 71, 713-723.  
17409690 T.Imai (2007).
[Hydrolysis by carboxylesterase and disposition of prodrug with ester moiety]
  Yakugaku Zasshi, 127, 611-619.  
17125253 D.J.Burkhart, B.L.Barthel, G.C.Post, B.T.Kalet, J.W.Nafie, R.K.Shoemaker, and T.H.Koch (2006).
Design, synthesis, and preliminary evaluation of doxazolidine carbamates as prodrugs activated by carboxylesterases.
  J Med Chem, 49, 7002-7012.  
16962139 S.Bencharit, C.C.Edwards, C.L.Morton, E.L.Howard-Williams, P.Kuhn, P.M.Potter, and M.R.Redinbo (2006).
Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1.
  J Mol Biol, 363, 201-214.
PDB codes: 2dqy 2dqz 2dr0 2h7c
16858120 T.Imai (2006).
Human carboxylesterase isozymes: catalytic properties and rational drug design.
  Drug Metab Pharmacokinet, 21, 173-185.  
16837465 Y.Bourne, Z.Radic, G.Sulzenbacher, E.Kim, P.Taylor, and P.Marchot (2006).
Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding.
  J Biol Chem, 281, 29256-29267.
PDB codes: 2h9y 2ha0 2ha2 2ha3 2ha4 2ha5 2ha6 2ha7
15601899 D.Gilham, M.Alam, W.Gao, D.E.Vance, and R.Lehner (2005).
Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.
  Mol Biol Cell, 16, 984-996.  
16167828 H.Huang, C.D.Fleming, K.Nishi, M.R.Redinbo, and B.D.Hammock (2005).
Stereoselective hydrolysis of pyrethroid-like fluorescent substrates by human and other mammalian liver carboxylesterases.
  Chem Res Toxicol, 18, 1371-1377.  
16160819 T.Ishikawa, A.Tamura, H.Saito, K.Wakabayashi, and H.Nakagawa (2005).
Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design.
  Naturwissenschaften, 92, 451-463.  
15948034 Z.Hu, X.Yang, P.C.Ho, E.Chan, S.Y.Chan, C.Xu, X.Li, Y.Z.Zhu, W.Duan, X.Chen, M.Huang, H.Yang, and S.Zhou (2005).
St. John's Wort modulates the toxicities and pharmacokinetics of CPT-11 (irinotecan) in rats.
  Pharm Res, 22, 902-914.  
12679808 S.Bencharit, C.L.Morton, Y.Xue, P.M.Potter, and M.R.Redinbo (2003).
Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme.
  Nat Struct Biol, 10, 349-356.
PDB codes: 1mx5 1mx9
15618752 S.R.Kim, T.Nakamura, Y.Saito, K.Sai, T.Nakajima, H.Saito, K.Shirao, H.Minami, A.Ohtsu, T.Yoshida, N.Saijo, S.Ozawa, and J.Sawada (2003).
Twelve novel single nucleotide polymorphisms in the CES2 gene encoding human carboxylesterase 2 (hCE-2).
  Drug Metab Pharmacokinet, 18, 327-332.  
12223468 M.Ito, U.Tchoua, M.Okamoto, and H.Tojo (2002).
Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis.
  J Biol Chem, 277, 43674-43681.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.