PDBsum entry 2flt

Hydrolase

PDB id: 2flt

Contents

Protein chain

117 a.a. *

Ligands

LAC

Waters ×60

* Residue conservation analysis

PDB id:

2flt

Name:

Hydrolase

Title:

The x-ray structure of the cis-3-chloroacrylic acid dehalogenase cis- caad inactivated with (r)-oxirane-2-carboxylate

Structure:

Cis-3-chloroacrylic acid dehalogenase. Chain: a. Engineered: yes

Source:

Coryneform bacterium. Organism_taxid: 1728. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PDB file)

Resolution:

2.10Å R-factor: 0.204 R-free: 0.226

Authors:

R.M.De Jong

Key ref:

R.M.de Jong et al. (2007). Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate. J Biol Chem, 282, 2440-2449. PubMed id: 17121835 DOI: 10.1074/jbc.M608134200

Date:

06-Jan-06 Release date: 21-Nov-06

Protein chain

Q6VPE5  (Q6VPE5_9CORY) -  Cis-3-chloroacrylic acid dehalogenase from coryneform bacterium

Seq: 150 a.a.

Struc: 117 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.8.1.- - ?????

DOI no: 10.1074/jbc.M608134200

J Biol Chem 282:2440-2449 (2007)

PubMed id: 17121835

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate.

R.M.de Jong, P.Bazzacco, G.J.Poelarends, W.H.Johnson, Y.J.Kim, E.A.Burks, H.Serrano, A.M.Thunnissen, C.P.Whitman, B.W.Dijkstra.

ABSTRACT

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.

Selected figure(s)

Figure 1.
FIGURE 1. Stereo views of (A) the monomeric and (B) the trimeric structure of cis-CaaD. The catalytic Pro-1 is shown in ball-and-stick (A) and Corey-Pauling-Koltun (B) representation. The figures were made using MOLSCRIPT and RASTER3D (24, 25).

Figure 2.
FIGURE 2. Detailed overview of the structure of native cis-CaaD and the final electron density of the covalent adduct in the structure of inactivated cis-CaaD. A, close-up stereo view of the active site of the native enzyme showing the interactions between the phosphate/sulfate ion and the two arginines (Arg-70 and Arg-73) and histidine (His-28). Residues are labeled by their chain color, except Pro-1, which is labeled in black. B, stereo view of the final 2F[o] – F[c] electron-density map contoured at 1.0 from XtalView (20) covering the proline and the covalently bound (R)-2-hydroxypropanoate adduct, clearly showing the tetrahedral conformations at the prolyl nitrogen and C2 of the adduct. The figures were prepared with MOLSCRIPT (A) (24) and RASTER3D (A and B) (24, 25).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 2440-2449) copyright 2007.

Figures were selected by the author.