4 research outputs found
Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5â˛-dAdo⢠âFree Radicalâ Is Never Free
Lysine 2,3-aminomutase (LAM) is a
radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme
and, like other members of this superfamily,
LAM utilizes radical-generating machinery comprising SAM anchored
to the unique Fe of a [4Fe-4S] cluster via a classical five-membered
N,O chelate ring. Catalysis is initiated by reductive cleavage of
the SAM SâC5Ⲡbond, which creates the highly reactive
5â˛-deoxyadenosyl radical (5â˛-dAdoâ˘), the same
radical generated by homolytic CoâC bond cleavage in B<sub>12</sub> radical enzymes. The SAM surrogate <i>S</i>-3â˛,4â˛-anhydroadenosyl-l-methionine (anSAM) can replace SAM as a cofactor in the isomerization
of l-ι-lysine to l-β-lysine by LAM,
via the stable allylic anhydroadenosyl radical (anAdoâ˘). Here
electron nuclear double resonance (ENDOR) spectroscopy of the anAdoâ˘
radical in the presence of <sup>13</sup>C, <sup>2</sup>H, and <sup>15</sup>N-labeled lysine completes the picture of how the active
site of LAM from <i>Clostridium subterminale</i> SB4 âtamesâ the 5â˛-dAdo⢠radical,
preventing it from carrying out harmful side reactions: this âfree
radicalâ in LAM is never free. The low steric demands of the
radical-generating [4Fe-4S]/SAM construct allow the substrate target
to bind adjacent to the SâC5Ⲡbond, thereby enabling
the 5â˛-dAdo⢠radical created by cleavage of this bond
to react with its partners by undergoing small motions, âź0.6
Ă
toward the target and âź1.5 Ă
overall, that are
controlled by tight van der Waals contact with its partners. We suggest
that the accessibility to substrate and ready control of the reactive
C5Ⲡradical, with âvan der Waals controlâ of
small motions throughout the catalytic cycle, is common within the
radical SAM enzyme superfamily and is a major reason why these enzymes
are the preferred means of initiating radical reactions in nature
Substrate-Dependent Cleavage Site Selection by Unconventional Radical <i>S</i>âAdenosylmethionine Enzymes in Diphthamide Biosynthesis
<i>S</i>-Adenosylmethionine (SAM) has a sulfonium ion
with three distinct C-S bonds. Conventional radical SAM enzymes use
a [4Fe-4S] cluster to cleave homolytically the C<sub>5â˛,adenosine</sub>-S bond of SAM to generate a 5â˛-deoxyadenosyl radical, which
catalyzes various downstream chemical reactions. Radical SAM enzymes
involved in diphthamide biosynthesis, such as Pyrococcus
horikoshii Dph2 (<i>Ph</i>Dph2) and yeast
Dph1-Dph2 instead cleave the C<sub>Îł,Met</sub>-S bond of methionine
to generate a 3-amino-3-carboxylpropyl radical. We here show radical
SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium
sulfur by changing the structure of SAM. With a decarboxyl SAM analogue
(dc-SAM), <i>Ph</i>Dph2 cleaves the C<sub>methyl</sub>-S
bond, forming 5â˛-deoxy-5â˛-(3-aminopropylthio) adenosine
(dAPTA, <b>1</b>). The methyl cleavage activity, like the cleavage
of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]<sup>+</sup> cluster. Electron-nuclear double resonance and mass spectroscopy
data suggests that mechanistically one of the S atoms in the [4Fe-4S]
cluster captures the methyl group from dc-SAM, forming a distinct
EPR-active intermediate, which can transfer the methyl group to nucleophiles
such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical
SAM enzyme can be tuned to cleave any one of the three bonds to the
sulfonium sulfur of SAM or analogues, and is the first demonstration
a radical SAM enzyme could switch from an Fe-based one electron transfer
reaction to a S-based two electron transfer reaction in a substrate-dependent
manner. This study provides an illustration of the versatile reactivity
of Fe-S clusters
Organometallic Complex Formed by an Unconventional Radical <i>S</i>âAdenosylmethionine Enzyme
<i>Pyrococcus horikoshii</i> Dph2 (<i>Ph</i>Dph2) is
an unusual radical <i>S</i>-adenosylmethionine
(SAM) enzyme involved in the first step of diphthamide biosynthesis.
It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl
(ACP) radical. To probe the reaction mechanism, we synthesized a SAM
analogue (SAM<sub>CA</sub>), in which the ACP group of SAM is replaced
with a 3-carboxyÂallyl group. SAM<sub>CA</sub> is cleaved by <i>Ph</i>Dph2, yielding a paramagnetic (<i>S</i> = 1/2)
species, which is assigned to a complex formed between the reaction
product, Îą-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster.
Electronânuclear double resonance (ENDOR) measurements with <sup>13</sup>C and <sup>2</sup>H isotopically labeled SAM<sub>CA</sub> support a Ď-complex between the CîťC double bond of
Îą-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S]
cluster. This is the first example of a radical SAM-related [4Fe-4S]<sup>+</sup> cluster forming an organometallic complex with an alkene,
shedding additional light on the mechanism of <i>Ph</i>Dph2
and expanding our current notions for the reactivity of [4Fe-4S] clusters
in radical SAM enzymes
Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme
Pyruvate formate-lyase
activating enzyme (PFL-AE) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that installs
a catalytically essential glycyl radical on pyruvate formate-lyase.
We show that PFL-AE binds a catalytically essential monovalent cation
at its active site, yet another parallel with B<sub>12</sub> enzymes,
and we characterize this cation site by a combination of structural,
biochemical, and spectroscopic approaches. Refinement of the PFL-AE
crystal structure reveals Na<sup>+</sup> as the most likely ion present
in the solved structures, and pulsed electron nuclear double resonance
(ENDOR) demonstrates that the same cation site is occupied by <sup>23</sup>Na in the solution state of the as-isolated enzyme. A SAM
carboxylate-oxygen is an M<sup>+</sup> ligand, and EPR and circular
dichroism spectroscopies reveal that both the site occupancy and the
identity of the cation perturb the electronic properties of the SAM-chelated
ironâsulfur cluster. ENDOR studies of the PFL-AE/[<sup>13</sup>C-methyl]-SAM complex show that the target sulfonium positioning
varies with the cation, while the observation of an isotropic hyperfine
coupling to the cation by ENDOR measurements establishes its intimate,
SAM-mediated interaction with the cluster. This monovalent cation
site controls enzyme activity: (i) PFL-AE in the absence of any simple
monovalent cations has littleâno activity; and (ii) among monocations,
going down Group 1 of the periodic table from Li<sup>+</sup> to Cs<sup>+</sup>, PFL-AE activity sharply maximizes at K<sup>+</sup>, with
NH<sub>4</sub><sup>+</sup> closely matching the efficacy of K<sup>+</sup>. PFL-AE is thus a type I M<sup>+</sup>-activated enzyme whose
M<sup>+</sup> controls reactivity by interactions with the cosubstrate,
SAM, which is bound to the catalytic ironâsulfur cluster