PDBsum entry 4u2c

Hydrolase

PDB id: 4u2c

Contents

Protein chain

233 a.a.

Ligands

SO4

Waters ×98

PDB id:

4u2c

Name:

Hydrolase

Title:

Crystal structure of dienelactone hydrolase a-6 variant (s7t, a24v, q35h, f38l, q110l, c123s, y145c, e199g and s208g) at 1.95 a resolution

Structure:

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

Source:

Pseudomonas sp.. Organism_taxid: 65741. Strain: b13. Gene: clcd. Expressed in: escherichia coli. Expression_system_taxid: 668369.

Resolution:

1.95Å R-factor: 0.181 R-free: 0.215

Authors:

J.L.Porter,C.A.Collyer,D.L.Ollis

Key ref:

J.L.Porter et al. (2015). Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search. Acs Chem Biol, 10, 611-621. PubMed id: 25419863 DOI: 10.1021/cb500809f

Date:

17-Jul-14 Release date: 10-Dec-14

Protein chain

P0A115  (CLCD_PSEKB) -  Carboxymethylenebutenolidase from Pseudomonas knackmussii (strain DSM 6978 / CCUG 54928 / LMG 23759 / B13)

Seq: 236 a.a.

Struc: 233 a.a.*

* PDB and UniProt seqs differ at 12 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.1.45 - carboxymethylenebutenolidase.
• Reaction: 2-(5-oxo-2,5-dihydrofuran-2-ylidene)acetate + H2O = 4-oxohex-2-enedioate + H⁺

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

Key reference

DOI no: 10.1021/cb500809f

Acs Chem Biol 10:611-621 (2015)

PubMed id: 25419863

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

J.L.Porter, P.L.Boon, T.P.Murray, T.Huber, C.A.Collyer, D.L.Ollis.

ABSTRACT

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.