4DNF

Crystal structure of the CFTR inhibitory factor Cif with the E153Q mutation adducted with the epibromohydrin hydrolysis intermediate

Summary for 4DNF

• Entry DOI: 10.2210/pdb4dnf/pdb
• Related: 3KD2 3KDA 3PI6 4DLN 4DM7 4DMC 4DMF 4DMH 4DMK 4DNO
• Descriptor: Putative hydrolase, (2S)-1-bromopropan-2-ol (3 entities in total)
• Functional Keywords: alpha beta hydrolase, epoxide hydrolase, covalently adducted with the epibromohydrin hydrolysis intermediate, secreted, hydrolase
• Biological source: Pseudomonas aeruginosa
• Total number of polymer chains: 4
• Total formula weight: 137210.82

Authors

Bahl, C.D.,Madden, D.R. (deposition date: 2012-02-08, release date: 2013-08-07, Last modification date: 2024-10-16)

Primary citation

Bahl, C.D.,Hvorecny, K.L.,Morisseau, C.,Gerber, S.A.,Madden, D.R.
Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif.
Biochemistry, 55:788-797, 2016

PubMed 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.

PubMed: 26752215
DOI: 10.1021/acs.biochem.5b01229

Experimental method

X-RAY DIFFRACTION (1.8 Å)