8 research outputs found
Investigation of Solvent Hydron Exchange in the Reaction Catalyzed by the Antibiotic Resistance Protein Cfr
Cfr
is a radical <i>S</i>-adenosylmethionine (RS) methylase
that appends methyl groups to C8 and C2 of adenosine 2503 in 23S rRNA.
Methylation of C8 confers resistance to several classes of antibiotics
that bind in or near the peptidyltransferase center of the bacterial
ribosome, including the synthetic antibiotic linezolid. The Cfr reaction
requires the action of five conserved cysteines, three of which ligate
a required [4Fe-4S] cluster cofactor. The two remaining cysteines
play a more intricate role in the reaction; one (Cys338) becomes transiently
methylated during catalysis. The function of the second (Cys105) has
not been rigorously established; however, in the related RlmN reaction,
it (Cys118) initiates resolution of a key protein–nucleic acid
cross-linked intermediate by abstracting the proton from the carbon
center (C2) undergoing methylation. We previously proposed that, unlike
RlmN, Cfr would utilize a polyprotic base during resolution of the
protein–nucleic acid cross-linked intermediate during C8 methylation
and, like RlmN, use a monoprotic base during C2 methylation. We based
this proposal on the fact that solvent hydrons could exchange into
the product during C8 methylation, but not during C2 methylation.
Herein, we show that Cys105 of Cfr has a function similar to that
of Cys118 of RlmN while methylating C8 of A2503 and provide evidence
for one molecule of water that is in close contact with it, which
provides the exchangeable protons during catalysis
Electrochemical Resolution of the [4Fe-4S] Centers of the AdoMet Radical Enzyme BtrN: Evidence of Proton Coupling and an Unusual, Low-Potential Auxiliary Cluster
The <i>S</i>-adenosylmethionine (AdoMet) radical superfamily
of enzymes includes over 113 500 unique members, each of which
contains one indispensable iron–sulfur (FeS) cluster that is
required to generate a 5′-deoxyadenosyl 5′-radical intermediate
during catalysis. Enzymes within several subgroups of the superfamily,
however, have been found to contain one or more additional FeS clusters.
While these additional clusters are absolutely essential for enzyme
activity, their exact roles in the function and/or mechanism of action
of many of the enzymes are at best speculative, indicating a need
to develop methods to characterize and study these clusters in more
detail. Here, BtrN, an AdoMet radical dehydrogenase that catalyzes
the two-electron oxidation of 2-deoxy-<i>scyllo</i>-inosamine
to amino-dideoxy-<i>scyllo</i>-inosose, an intermediate
in the biosynthesis of 2-deoxystreptamine antibiotics, is examined
through direct electrochemistry, where the potential of both its AdoMet
radical and auxiliary [4Fe-4S] clusters can be measured simultaneously.
We find that the AdoMet radical cluster exhibits a midpoint potential
of −510 mV, while the auxiliary cluster exhibits a midpoint
potential of −765 mV, to our knowledge the lowest [4Fe-4S]<sup>2+/+</sup> potential to be determined to date. The impact of AdoMet
binding and the pH dependence of catalysis are also quantitatively
observed. These data show that direct electrochemical methods can
be used to further elucidate the chemistry of the burgeoning AdoMet
radical superfamily in the future
NosN, a Radical <i>S</i>‑Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide
Nosiheptide, a member of the <i>e</i> series of macrocyclic thiopeptide natural products, contains
a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety
connected to Glu6 and Cys8 of the thiopeptide backbone via ester and
thioester linkages, respectively. Herein, we show that NosN, a predicted
class C radical <i>S</i>-adenosylmethionine (SAM) methylase,
catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic
acid linked to Cys8 of a synthetic substrate surrogate as well as
the formation of the ester linkage between Glu6 and the nascent C4
methylene moiety of DMIA. In contrast to previous studies that indicated
that 5′-methylthioadenosine is the immediate methyl donor in
the reaction, in our studies, SAM itself plays this role, giving rise
to <i>S</i>-adenosylhomocysteine as a coproduct of the reaction
Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK
Nosiheptide (NOS) is a highly modified
thiopeptide antibiotic that
displays formidable in vitro activity against a variety of Gram-positive
bacteria. In addition to a central hydroxypyridine ring, NOS contains
several other modifications, including multiple thiazole rings, dehydro-amino
acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety
is required for NOS efficacy and is synthesized from l-tryptophan
in a series of reactions that have not been fully elucidated. Herein,
we describe the role of NosJ, the product of an unannotated gene in
the biosynthetic operon for NOS, as an acyl carrier protein that delivers
3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI
as the enzyme responsible for catalyzing the ATP-dependent activation
of MIA and MIA’s attachment to the phosphopantetheine moiety
of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from
NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray
crystal structure of NosK, solved to 2.3 Ã… resolution, reveals
that the protein is an α/β-fold hydrolase. Ser102 interacts
with Glu210 and His234 to form a catalytic triad located at the bottom
of an open cleft that is large enough to accommodate the thiopeptide
framework
Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme
We
report on the protonation state of <i>Helicobacter pylori</i> catalase compound II. UV/visible, Mössbauer, and X-ray absorption
spectroscopies have been used to examine the intermediate from pH
5 to 14. We have determined that HPC-II exists in an ironÂ(IV) hydroxide
state up to pH 11. Above this pH, the ironÂ(IV) hydroxide complex transitions
to a new species (p<i>K</i><sub>a</sub> = 13.1) with Mössbauer
parameters that are indicative of an ironÂ(IV)-oxo intermediate. Recently,
we discussed a role for an elevated compound II p<i>K</i><sub>a</sub> in diminishing the compound I reduction potential. This
has the effect of shifting the thermodynamic landscape toward the
two-electron chemistry that is critical for catalase function. In
catalase, a diminished potential would increase the selectivity for
peroxide disproportionation over off-pathway one-electron chemistry,
reducing the buildup of the inactive compound II state and reducing
the need for energetically expensive electron donor molecules
Structure of Quinolinate Synthase from <i>Pyrococcus horikoshii</i> in the Presence of Its Product, Quinolinic Acid
Quinolinic
acid (QA) is a common intermediate in the biosynthesis
of nicotinamide adenine dinucleotide (NAD<sup>+</sup>) and its derivatives
in all organisms that synthesize the molecule <i>de novo</i>. In most prokaryotes, it is formed from the condensation of dihydroxyacetone
phosphate (DHAP) and aspartate-enamine by the action of quinolinate
synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a
unique, non-cysteinyl-ligated, iron ion (Fe<sub>a</sub>), which is
proposed to bind the hydroxyl group of a postulated intermediate in
the last step of the reaction to facilitate a dehydration. However,
direct evidence for this role in catalysis has yet to be provided.
Herein, we present the structure of NadA in the presence of the product
of its reaction, QA. We find that N1 and the C7 carboxylate group
of QA ligate to Fe<sub>a</sub> in a bidentate fashion, which is confirmed
by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding
mode would place the C5 hydroxyl group of the postulated final intermediate
distal to Fe<sub>a</sub> and virtually incapable of coordinating to
it. The structure shows that three strictly conserved amino acids,
Glu198, Tyr109, and Tyr23, are in close proximity to the bound product.
Substitution of these amino acids with Gln, Phe, and Phe, respectively,
leads to complete loss of activity
Characterization of a Cross-Linked Protein–Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN
RlmN
and Cfr are methyltransferases/methylsynthases that belong
to the radical <i>S</i>-adenosylmethionine superfamily of
enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of
23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide,
and will subsequently catalyze C2 methylation if the site is unmethylated.
A key feature of the unusual mechanisms of catalysis proposed for
these enzymes is the attack of a methylene radical, derived from a
methylcysteine residue, onto the carbon center undergoing methylation
to generate a paramagnetic protein–nucleic acid cross-linked
species. This species has been thoroughly characterized during Cfr-dependent
C8 methylation, but does not accumulate to detectible levels in RlmN-dependent
C2 methylation. Herein, we show that inactive C118S/A variants of
RlmN accumulate a substrate-derived paramagnetic species. Characterization
of this species by electron paramagnetic resonance spectroscopy in
concert with strategic isotopic labeling shows that the radical is
delocalized throughout the adenine ring of A2503, although predominant
spin density is on N1 and N3. Moreover, <sup>13</sup>C hyperfine interactions
between the radical and the methylene carbon of the formerly [<i>methyl</i>-<sup>13</sup>C]ÂCys355 residue show that the radical
species exists in a covalent cross-link between the protein and the
nucleic acid substrate. X-ray structures of RlmN C118A show that,
in the presence of SAM, the substitution does not alter the active
site structure compared to that of the wild-type enzyme. Together,
these findings have new mechanistic implications for the role(s) of
C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement
of the residue in resolution of the cross-linked species via a radical
mediated process
Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients
We present compelling
evidence for the existence of an extended
innate viperin-dependent pathway, which provides crucial evidence
for an adaptive response to viral agents, such as SARS-CoV-2. We show
the in vivo biosynthesis of a family of novel endogenous cytosine
metabolites with potential antiviral activities. Two-dimensional nuclear
magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system
motif, indicating the presence of an extended panel of urinary metabolites
during the acute viral replication phase. Mass spectrometry additionally
enabled the characterization and quantification of the most abundant
serum metabolites, showing the potential diagnostic value of the compounds
for viral infections. In total, we unveiled ten nucleoside (cytosine-
and uracil-based) analogue structures, eight of which were previously
unknown in humans allowing us to propose a new extended viperin
pathway for the innate production of antiviral compounds.
The molecular structures of the nucleoside analogues and their correlation
with an array of serum cytokines, including IFN-α2, IFN-γ,
and IL-10, suggest an association with the viperin enzyme contributing
to an ancient endogenous innate immune defense mechanism against viral
infection