4XBT

Crystal Structure of the L74F/M78F/L103V/L114V/I116V/F139V/L147V mutant of LEH complexed with (S,S)-cyclohexanediol

Summary for 4XBT

• Entry DOI: 10.2210/pdb4xbt/pdb
• Related: 4XBX 4XBY 4XDV 4XDW
• Descriptor: Limonene-1,2-epoxide hydrolase, (1S,2S)-cyclohexane-1,2-diol, CITRATE ANION, ... (4 entities in total)
• Functional Keywords: epoxide hydrolase, product complex, hydrolase
• Biological source: Rhodococcus erythropolis
• Total number of polymer chains: 4
• Total formula weight: 69847.74

Authors

Kong, X.D.,Sun, Z.,Lonsdale, R.,Xu, J.H.,Reetz, M.T.,Zhou, J. (deposition date: 2014-12-17, release date: 2015-07-15, Last modification date: 2023-11-08)

Primary citation

Sun, Z.,Lonsdale, R.,Kong, X.D.,Xu, J.H.,Zhou, J.,Reetz, M.T.
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, 2015

PubMed 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.

PubMed: 25891639
DOI: 10.1002/anie.201501809

Experimental method

X-RAY DIFFRACTION (1.7 Å)