PDBsum entry 5eht

Hydrolase

PDB id: 5eht

Contents

Protein chain

253 a.a.

Ligands

GOL ×6

Metals

_ZN ×2

Waters ×247

PDB id:

5eht

Name:

Hydrolase

Title:

Indirect contributions of mutations underlie optimization of new enzyme function

Structure:

N-acyl homoserine lactonase. Chain: a. Synonym: ahl-lactonase,homoserine lactone lactonase. Engineered: yes. Mutation: yes

Source:

Bacillus thuringiensis. Organism_taxid: 1428. Gene: aiia. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.29Å R-factor: 0.128 R-free: 0.168

Authors:

C.J.Jackson,N.-S.Hong

Key ref:

G.Yang et al. (2016). Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects. Biochemistry, 55, 4583-4593. PubMed id: 27444875 DOI: 10.1021/acs.biochem.6b00561

Date:

28-Oct-15 Release date: 07-Sep-16

Protein chain

A3FJ64  (AHLL_BACTU) -  N-acyl homoserine lactonase from Bacillus thuringiensis

Seq: 250 a.a.

Struc: 253 a.a.*

* PDB and UniProt seqs differ at 8 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.1.81 - quorum-quenching N-acyl-homoserine lactonase.
• Reaction: an N-acyl-L-homoserine lactone + H2O = an N-acyl-L-homoserine + H⁺

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

Added reference

DOI no: 10.1021/acs.biochem.6b00561

Biochemistry 55:4583-4593 (2016)

PubMed id: 27444875

Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

G.Yang, N.Hong, F.Baier, C.J.Jackson, N.Tokuriki.

ABSTRACT

How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.