PDBsum entry: 1zj5

Hydrolase

PDB id: 1zj5

Contents

Protein chain

233 a.a. *

Ligands

SO4 ×2

GOL

Waters ×124

* Residue conservation analysis

PDB id:

1zj5

Name:

Hydrolase

Title:

Crystal structure analysis of the dienelactone hydrolase mut c123s, a134s, s208g, a229v, k234r) bound with the pms moiet protease inhibitor, phenylmethylsulfonyl fluoride (pmsf)- 1

Structure:

Carboxymethylenebutenolidase. Chain: a. Synonym: dienelactone hydrolase. Engineered: yes. Mutation: yes

Source:

Pseudomonas putida. Organism_taxid: 303. Gene: clcd. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.70Å R-factor: 0.184 R-free: 0.210

Authors:

H.-K.Kim,J.-W.Liu,P.D.Carr,D.L.Ollis

Key ref:

H.K.Kim et al. (2005). Following directed evolution with crystallography: structural changes observed in changing the substrate specificity of dienelactone hydrolase. Acta Crystallogr D Biol Crystallogr, 61, 920-931. PubMed id: 15983415 DOI: 10.1107/S0907444905009042

Date:

28-Apr-05 Release date: 05-Jul-05

Protein chain

P0A114  (CLCD_PSEPU) -  Carboxymethylenebutenolidase

Seq: 236 a.a.

Struc: 233 a.a.*

* PDB and UniProt seqs differ at 7 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.1.45 - Carboxymethylenebutenolidase.
• Reaction: 4-carboxymethylenebut-2-en-4-olide + H₂O = 4-oxohex-2-enedioate

4-carboxymethylenebut-2-en-4-olide (Bound ligand (Het Group name = GOL) matches with 45.45% similarity) + H(2)O = 4-oxohex-2-enedioate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: aromatic compound catabolic process (1 term)
• Biochemical function: hydrolase activity (3 terms)

Added reference

DOI no: 10.1107/S0907444905009042

Acta Crystallogr D Biol Crystallogr 61:920-931 (2005)

PubMed id: 15983415

Following directed evolution with crystallography: structural changes observed in changing the substrate specificity of dienelactone hydrolase.

H.K.Kim, J.W.Liu, P.D.Carr, D.L.Ollis.

ABSTRACT

The enzyme dienelactone hydrolase (DLH) has undergone directed evolution to produce a series of mutant proteins that have enhanced activity towards the non-physiological substrates alpha-naphthyl acetate and p-nitrophenyl acetate. In terms of steady-state kinetics, the mutations caused a drop in the K(m) for the hydrolysis reaction with these two substrates. For the best mutant, there was a 5.6-fold increase in k(cat)/K(m) for the hydrolysis of alpha-naphthyl acetate and a 3.6-fold increase was observed for p-nitrophenyl acetate. For alpha-naphthyl acetate the pre-steady-state kinetics revealed that the rate constant for the formation of the covalent intermediate had increased. The mutations responsible for the rate enhancements map to the active site. The structures of the starting and mutated proteins revealed small changes in the protein owing to the mutations, while the structures of the same proteins with an inhibitor co-crystallized in the active site indicated that the mutations caused significant changes in the way the mutated proteins recognized the substrates. Within the active site of the mutant proteins, the inhibitor was rotated by about 180 degrees with respect to the orientation found in the starting enzyme. This rotation of the inhibitor caused the displacement of a large section of a loop on one side of the active site. Residues that could stabilize the transition state for the reaction were identified.

Selected figure(s)

Figure 4.
Figure 4 Schematic diagrams showing the active sites of (a) DLH-C1 with only the C123S mutation, (b) DLH-G2-06 with the C123S and E36D mutations and (c) DLH-G4-113 with the C1232S, E36D, R105H, G211D and K234N mutations. Arrows indicate the relative orientations of discreetly disordered side chains Asp36 and Arg206. (d) DLH-G3-16 with the C123S, E36D, A134S, S208G, V229V and K234R mutations.

Figure 5.
Figure 5 Diagram of the refined structures showing the active site and the loop region (residues 165-185, C^ [alpha] only) of DLH-C1 (grey bonds) DLH-C1-PMS (green bonds), G2-06-PMS (lavender bonds) and G3-16-PMS (cyan bonds). The structures of G2-06-PMS and G3-16-PMS were overlaid onto DLH-C1-PMS using all residues with the program LSQKAB (Kabsch, 1976 [Kabsch, W. (1976). Acta Cryst. A32, 922-923.]-[bluearr.gif] ).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2005, 61, 920-931) copyright 2005.

Figures were selected by an automated process.