17 research outputs found
Redesign of Substrate Specificity and Identification of the Aminoglycoside Binding Residues of Eis from Mycobacterium tuberculosis
The upsurge in drug-resistant tuberculosis
(TB) is an emerging
global problem. The increased expression of the enhanced intracellular
survival (Eis) protein is responsible for the clinical resistance
to aminoglycoside (AG) antibiotics of Mycobacterium
tuberculosis. Eis from M. tuberculosis (Eis_<i>Mtb</i>) and M. smegmatis (Eis_<i>Msm</i>) function as acetyltransferases capable
of acetylating multiple amines of many AGs; however, these Eis homologues
differ in AG substrate preference and in the number of acetylated
amine groups per AG. The AG binding cavity of Eis_<i>Mtb</i> is divided into two narrow channels, whereas Eis_<i>Msm</i> contains one large cavity. Five bulky residues lining one of the
AG binding channels of Eis_<i>Mtb</i>, His119, Ile268, Trp289,
Gln291, and Glu401, have significantly smaller counterparts in Eis_<i>Msm</i>, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively.
To identify the residue(s) responsible for AG binding in Eis_<i>Mtb</i> and for the functional differences from Eis_<i>Msm</i>, we have generated single, double, triple, quadruple,
and quintuple mutants of these residues in Eis_<i>Mtb</i> by mutating them into their Eis_<i>Msm</i> counterparts,
and we tested their acetylation activity with three structurally diverse
AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We
show that penultimate C-terminal residue Glu401 plays a critical role
in the overall activity of Eis_<i>Mtb</i>. We also demonstrate
that the identities of residues Ile268, Trp289, and Gln291 (in Eis_<i>Mtb</i> nomenclature) dictate the differences between the acetylation
efficiencies of Eis_<i>Mtb</i> and Eis_<i>Msm</i> for KAN and PAR. Finally, we show that the mutation of Trp289 in
Eis_<i>Mtb</i> into Ala plays a role in APR acetylation
Synthesis and Evaluation of Hetero- and Homodimers of Ribosome-Targeting Antibiotics: Antimicrobial Activity, in Vitro Inhibition of Translation, and Drug Resistance
In
this study, we describe the synthesis of a full set of homo-
and heterodimers of three intact structures of different ribosome-targeting
antibiotics: tobramycin, clindamycin, and chloramphenicol. Several
aspects of the biological activity of the dimeric structures were
evaluated including antimicrobial activity, inhibition of in vitro
bacterial protein translation, and the effect of dimerization on the
action of several bacterial resistance mechanisms that deactivate
tobramycin and chloramphenicol. This study demonstrates that covalently
linking two identical or different ribosome-targeting antibiotics
may lead to (i) a broader spectrum of antimicrobial activity, (ii)
improved inhibition of bacterial translation properties compared to
that of the parent antibiotics, and (iii) reduction in the efficacy
of some drug-modifying enzymes that confer high levels of resistance
to the parent antibiotics from which the dimers were derived
Methylated <i>N</i><sup>ω</sup>‑Hydroxy‑l‑arginine Analogues as Mechanistic Probes for the Second Step of the Nitric Oxide Synthase-Catalyzed Reaction
Nitric
oxide synthase (NOS) catalyzes the conversion of l-arginine
to l-citrulline through the intermediate <i>N</i><sup>ω</sup>-hydroxy-l-arginine (NHA), producing
nitric oxide, an important mammalian signaling molecule. Several disease
states are associated with improper regulation of nitric oxide production,
making NOS a therapeutic target. The first step of the NOS reaction
has been well-characterized and is presumed to proceed through a compound
I heme species, analogous to the cytochrome P450 mechanism. The second
step, however, is enzymatically unprecedented and is thought to occur
via a ferric peroxo heme species. To gain insight into the details
of this unique second step, we report here the synthesis of NHA analogues
bearing guanidinium methyl or ethyl substitutions and their investigation
as either inhibitors of or alternate substrates for NOS. Radiolabeling
studies reveal that <i>N</i><sup>ω</sup>-methoxy-l-arginine, an alternative NOS substrate, produces citrulline,
nitric oxide, and methanol. On the basis of these results, we propose
a mechanism for the second step of NOS catalysis in which a methylated
nitric oxide species is released and is further metabolized by NOS.
Crystal structures of our NHA analogues bound to nNOS have been determined,
revealing the presence of an active site water molecule only in the
presence of singly methylated analogues. Bulkier analogues displace
this active site water molecule; a different mechanism is proposed
in the absence of the water molecule. Our results provide new insights
into the steric and stereochemical tolerance of the NOS active site
and substrate capabilities of NOS
Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications
Cysteine <i>S</i>-nitrosation
and <i>S</i>-sulfination
are naturally occurring post-translational modifications (PTMs) on
proteins induced by physiological signals and redox stress. Here we
demonstrate that sulfinic acids and nitrosothiols react to form a
stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked
probes to enrich and annotate hundreds of endogenous <i>S</i>-nitrosated proteins. In physiological buffers, sulfinic acids do
not react with iodoacetamide or disulfides, enabling selective alkylation
of free thiols and site-specific analysis of <i>S</i>-nitrosation.
In parallel, <i>S</i>-nitrosothiol-linked probes enable
enrichment and detection of endogenous <i>S</i>-sulfinated
proteins, confirming that a single sulfinic acid can react with a
nitrosothiol to form a thiosulfonate linkage. Using this approach,
we find that hydrogen peroxide addition increases <i>S</i>-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53
are fully oxidized to sulfonic acids. Comparative gel-based analysis
of different mouse tissues reveals distinct profiles for both <i>S</i>-nitrosation and <i>S</i>-sulfination. Quantitative
proteomic analysis demonstrates that both <i>S</i>-nitrosation
and <i>S</i>-sulfination are widespread, yet exhibit enhanced
occupancy on select proteins, including thioredoxin, peroxiredoxins,
and other validated redox active proteins. Overall, we present a direct,
bidirectional method to profile select redox cysteine modifications
based on the unique nucleophilicity of sulfinic acids
Redesign of Substrate Specificity and Identification of the Aminoglycoside Binding Residues of Eis from Mycobacterium tuberculosis
Improved Synthesis of Chiral Pyrrolidine Inhibitors and Their Binding Properties to Neuronal Nitric Oxide Synthase
Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)
Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)
Post-translational <i>S</i>-palmitoylation directs the
trafficking and membrane localization of hundreds of cellular proteins,
often involving a coordinated palmitoylation cycle that requires both
protein acyl transferases (PATs) and acyl protein thioesterases (APTs)
to actively redistribute <i>S</i>-palmitoylated proteins
toward different cellular membrane compartments. This process is necessary
for the trafficking and oncogenic signaling of <i>S</i>-palmitoylated
Ras isoforms, and potentially many peripheral membrane proteins. The
depalmitoylating enzymes APT1 and APT2 are separately conserved in
all vertebrates, suggesting unique functional roles for each enzyme.
The recent discovery of the APT isoform-selective inhibitors ML348
and ML349 has opened new possibilities to probe the function of each
enzyme, yet it remains unclear how each inhibitor achieves orthogonal
inhibition. Herein, we report the high-resolution structure of human
APT2 in complex with ML349 (1.64 Å), as well as the complementary
structure of human APT1 bound to ML348 (1.55 Å). Although the
overall peptide backbone structures are nearly identical, each inhibitor
adopts a distinct conformation within each active site. In APT1, the
trifluoromethyl group of ML348 is positioned above the catalytic triad,
but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with
active site resident waters to indirectly engage the catalytic triad
and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed
several differing residues surrounding the active site that serve
as critical gatekeepers for isoform accessibility and dynamics. Structural
and biochemical analysis suggests the inhibitors occupy a putative
acyl-binding region, establishing the mechanism for isoform-specific
inhibition, hydrolysis of acyl substrates, and structural orthogonality
important for future probe development