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PDBsum entry 4dnf
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References listed in PDB file
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Key reference
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Title
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Visualizing the mechanism of epoxide hydrolysis by the bacterial virulence enzyme cif.
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Authors
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C.D.Bahl,
K.L.Hvorecny,
C.Morisseau,
S.A.Gerber,
D.R.Madden.
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Ref.
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Biochemistry, 2016,
55,
788-797.
[DOI no: ]
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PubMed id
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Abstract
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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.
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