5 research outputs found
Structures and Mechanisms of the Non-Heme Fe(II)- and 2‑Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist
The ethylene-forming enzyme (EFE)
from <i>Pseudomonas syringae</i> pv. <i>phaseolicola</i> PK2 is a member of the mononuclear
nonheme FeÂ(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily.
EFE converts 2OG into ethylene plus three CO<sub>2</sub> molecules
while also catalyzing the C5 hydroxylation of l-arginine
(l-Arg) driven by the oxidative decarboxylation of 2OG to
form succinate and CO<sub>2</sub>. Here we report 11 X-ray crystal
structures of EFE that provide insight into the mechanisms of these
two reactions. Binding of 2OG in the absence of l-Arg resulted
in predominantly monodentate metal coordination, distinct from the
typical bidentate metal-binding species observed in other family members.
Subsequent addition of l-Arg resulted in compression of the
active site, a conformational change of the carboxylate side chain
metal ligand to allow for hydrogen bonding with the substrate, and
creation of a twisted peptide bond involving this carboxylate and
the following tyrosine residue. A reconfiguration of 2OG achieves
bidentate metal coordination. The dioxygen binding site is located
on the metal face opposite to that facing l-Arg, thus requiring
reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward
the metal may hinder such a ferryl flip and promote ethylene formation.
Extensive site-directed mutagenesis studies supported the importance
of this phenylalanine and confirmed the essential residues used for
substrate binding and catalysis. The structural and functional characterization
described here suggests that conversion of 2OG to ethylene, atypical
among FeÂ(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate
metal ligand, a resulting twisted peptide bond, and the off-line geometry
for dioxygen coordination
Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism
Lactate racemase (LarA) of <i>Lactobacillus plantarum</i> contains a novel organometallic
cofactor with nickel coordinated
to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic
acid mononucleotide, but its function in the enzyme mechanism has
not been elucidated. This study presents direct evidence that the
nickel-pincer cofactor facilitates a proton-coupled hydride transfer
(PCHT) mechanism during LarA-catalyzed lactate racemization. No signal
was detected by electron paramagnetic resonance spectroscopy for LarA
in the absence or presence of substrate, consistent with a +2 metal
oxidation state and inconsistent with a previously proposed proton-coupled
electron transfer mechanism. Pyruvate, the predicted intermediate
for a PCHT mechanism, was observed in quenched solutions of LarA.
A normal substrate kinetic isotope effect (<i>k</i><sub>H</sub>/<i>k</i><sub>D</sub> of 3.11 ± 0.17) was established
using 2-α-<sup>2</sup>H-lactate, further supporting a PCHT mechanism.
UV–visible spectroscopy revealed a lactate-induced perturbation
of the cofactor spectrum, notably increasing the absorbance at 340
nm, and demonstrated an interaction of the cofactor with the inhibitor
sulfite. A crystal structure of LarA provided greater resolution (2.4
Ă…) than previously reported and revealed sulfite binding to the
pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride
reduction during a PCHT catalytic cycle. Finally, computational modeling
supports hydride transfer to the cofactor at the C4 position or to
the nickel atom, but with formation of a nickel-hydride species requiring
dissociation of the His200 metal ligand. In aggregate, these studies
provide compelling evidence that the nickel-pincer cofactor acts by
a PCHT mechanism
Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections
Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible
for a wide range of systemic infections. Since its propensity to form
biofilms in vivo poses formidable challenges for
both detection and treatment, tools that can be used to specifically
image S. aureus biofilms are highly
valuable for clinical management. Here, we describe the development
of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because
this enzyme lacks homologues in other bacteria, it is an ideal target
for selective imaging of S. aureus infections.
Using X-ray crystallography, direct cell labeling, and mouse models
of infection, we demonstrate that oxadiazolone-based probes enable
specific labeling of S. aureus bacteria
through the direct covalent modification of the FphE active site serine.
These results demonstrate the utility of the oxadizolone electrophile
for activity-based probes and validate FphE as a target for the development
of imaging contrast agents for the rapid detection of S. aureus infections
Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections
Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible
for a wide range of systemic infections. Since its propensity to form
biofilms in vivo poses formidable challenges for
both detection and treatment, tools that can be used to specifically
image S. aureus biofilms are highly
valuable for clinical management. Here, we describe the development
of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because
this enzyme lacks homologues in other bacteria, it is an ideal target
for selective imaging of S. aureus infections.
Using X-ray crystallography, direct cell labeling, and mouse models
of infection, we demonstrate that oxadiazolone-based probes enable
specific labeling of S. aureus bacteria
through the direct covalent modification of the FphE active site serine.
These results demonstrate the utility of the oxadizolone electrophile
for activity-based probes and validate FphE as a target for the development
of imaging contrast agents for the rapid detection of S. aureus infections
Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in <i>Staphylococcus aureus</i> Support a Role in Bacterial Stress Response
The development of
new treatment options for bacterial
infections
requires access to new targets for antibiotics and antivirulence strategies.
Chemoproteomic approaches are powerful tools for profiling and identifying
novel druggable target candidates, but their functions often remain
uncharacterized. Previously, we used activity-based protein profiling
in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding
hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred
to as yvaK in Bacillus subtilis)
at the molecular and cellular level. First, phenotypic characterization
of fphH-deficient transposon mutants revealed phenotypes
during growth under nutrient deprivation, biofilm formation, and intracellular
survival. Biochemical and structural investigations revealed that
FphH acts as an esterase and lipase based on a fold well suited to
act on a small to long hydrophobic unbranched lipid group within its
substrate and can be inhibited by active site-targeting oxadiazoles.
Prompted by a previous observation that fphH expression
was upregulated in response to fusidic acid, we found that FphH can
deacetylate this ribosome-targeting antibiotic, but the lack of FphH
function did not infer major changes in antibiotic susceptibility.
In conclusion, our results indicate a functional role of this hydrolase
in S. aureus stress responses, and hypothetical functions
connecting FphH with components of the ribosome rescue system that
are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate
the development of specific FphH inhibitors and probes to elucidate
its physiological role and validity as a drug target