PDBsum entry 2w3x

Hydrolase

PDB id: 2w3x

Contents

Protein chains

(+ 0 more) 142 a.a. *

Ligands

PO4 ×2

JEF

POP ×2

GOL ×2

Waters ×526

* Residue conservation analysis

PDB id:

2w3x

Name:

Hydrolase

Title:

Crystal structure of a bifunctional hotdog fold thioesterase in enediyne biosynthesis, cale7

Structure:

Cale7. Chain: a, b, c, d, e, f. Fragment: residues 1-147. Engineered: yes

Source:

Micromonospora echinospora. Organism_taxid: 1877. Strain: calichensis. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.75Å R-factor: 0.196 R-free: 0.235

Authors:

M.Kotaka,R.Kong,I.Qureshi,Q.S.Ho,H.Sun,C.W.Liew,L.P.Goh,P.Cheung, Y.Mu,J.Lescar,Z.X.Liang

Key ref:

M.Kotaka et al. (2009). Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis. J Biol Chem, 284, 15739-15749. PubMed id: 19357082 DOI: 10.1074/jbc.M809669200

Date:

17-Nov-08 Release date: 07-Apr-09

Protein chains

Q8KNG2  (Q8KNG2_MICEC) -  CalE7 from Micromonospora echinospora

Seq: 148 a.a.

Struc: 142 a.a.

DOI no: 10.1074/jbc.M809669200

J Biol Chem 284:15739-15749 (2009)

PubMed id: 19357082

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis.

M.Kotaka, R.Kong, I.Qureshi, Q.S.Ho, H.Sun, C.W.Liew, L.P.Goh, P.Cheung, Y.Mu, J.Lescar, Z.X.Liang.

ABSTRACT

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-A crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg(37) plays a crucial role in the hydrolysis of the thioester bond, and that Tyr(29) and a hydrogen-bonded water network assist the decarboxylation of the beta-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.

Selected figure(s)

Figure 1.
Calicheamicin and its biosynthesis. A, structure of calicheamicin γ′[1] with the incorporated acetate units in the 10-membered enediyne moiety highlighted in bold sticks. B, early steps of the biosynthetic pathway of the 10-membered enediyne as proposed by Kong et al. (13). The incorporated acetate units are highlighted in bold sticks with the configuration of the double bonds in the intermediates arbitrarily assigned. (AT, acyl transferase; KS, ketoacyl synthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; and PPTase, phosphopantetheinyl transferase.).

Figure 3.
Sequence and structure of CalE7. A, sequence alignment of CalE7 with SgcE10 and the hypothetical protein (AQ1494) from A. aeolicus (PDB code 2EGI). Strictly conserved residues are highlighted in red, and partially conserved residues are yellow. The residues mutated in the present study are indicated by blue asterisks. B, representation of the CalE7 monomer. The protein secondary structures elements are labeled and colored from blue at the N terminus to red at the C terminus. C, schematic representation of the CalE7 tetramer showing the Jeffamine fragment (sticks) binding site between two subunits. Subunits are colored gray and cyan for one dimer and dark blue and dark gray for the other. The β5–α3 loops at the active sites is colored red. The inset shows a close-up view of the Jeffamine binding site superposed with 4-hydroxybenzoyl-CoA-bound 4-BHT (PDB code: 1LO9). D, simulated annealing difference Fourier map with coefficients |F[obs]| − |F[calc]| and phases calculated from the protein model with atoms from the Jeffamine fragment omitted and contoured at a level of 3σ.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 15739-15749) copyright 2009.

Figures were selected by the author.