PDBsum entry 4dnf

Hydrolase

PDB id: 4dnf

Contents

Protein chains

296 a.a.

Ligands

EBH ×4

Waters ×829

PDB id:

4dnf

Name:

Hydrolase

Title:

Crystal structure of the cftr inhibitory factor cif with the e153q mutation adducted with the epibromohydrin hydrolysis intermediate

Structure:

Putative hydrolase. Chain: a, b, c, d. Fragment: cif (unp residues 25-319). Engineered: yes. Mutation: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 208963. Strain: ucbpp-pa14. Gene: pa14_26090. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.80Å R-factor: 0.191 R-free: 0.224

Authors:

C.D.Bahl,D.R.Madden

Key ref:

C.D.Bahl et al. (2016). Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif. Biochemistry, 55, 788-797. PubMed id: 26752215 DOI: 10.1021/acs.biochem.5b01229

Date:

08-Feb-12 Release date: 07-Aug-13

Protein chains

A0A0H2ZD27  (A0A0H2ZD27_PSEAB) -  Putative hydrolase from Pseudomonas aeruginosa (strain UCBPP-PA14)

Seq: 319 a.a.

Struc: 296 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1021/acs.biochem.5b01229

Biochemistry 55:788-797 (2016)

PubMed id: 26752215

Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif.

C.D.Bahl, K.L.Hvorecny, C.Morisseau, S.A.Gerber, D.R.Madden.

ABSTRACT

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.