PDBsum entry 4xby

Hydrolase

PDB id: 4xby

Contents

Protein chains

(+ 2 more) 144 a.a.

Ligands

3ZS ×4

Waters ×596

PDB id:

4xby

Name:

Hydrolase

Title:

Crystal structure of the l74f/m78v/i80v/l114f mutant of leh complexed with cyclopentene oxide

Structure:

Limonene-1,2-epoxide hydrolase. Chain: c, d, a, b, e, f, g, h. Fragment: unp residues 5-149. Engineered: yes. Mutation: yes

Source:

Rhodococcus erythropolis. Organism_taxid: 1833. Gene: lima. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.30Å R-factor: 0.214 R-free: 0.269

Authors:

X.D.Kong,Z.Sun,J.H.Xu,M.T.Reetz,J.Zhou

Key ref:

Z.Sun et al. (2015). Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution. Angew Chem Int Ed Engl, 54, 12410-12415. PubMed id: 25891639 DOI: 10.1002/anie.201501809

Date:

17-Dec-14 Release date: 15-Jul-15

Protein chains

Q9ZAG3  (LIMA_RHOER) -  Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis

Seq: 149 a.a.

Struc: 144 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.3.2.8 - limonene-1,2-epoxide hydrolase.
• Reaction: limonene 1,2-epoxide + H2O = limonene-1,2-diol

limonene 1,2-epoxide + H2O (Bound ligand (Het Group name = 3ZS) matches with 54.55% similarity) = limonene-1,2-diol

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

Added reference

DOI no: 10.1002/anie.201501809

Angew Chem Int Ed Engl 54:12410-12415 (2015)

PubMed id: 25891639

Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.

Z.Sun, R.Lonsdale, X.D.Kong, J.H.Xu, J.Zhou, M.T.Reetz.

ABSTRACT

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.