7 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
Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor
ABSTRACTAcetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and MĂśssbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design
Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation
Nature utilizes [FeFe]-hydrogenase
enzymes to catalyze the interconversion
between H<sub>2</sub> and protons and electrons. Catalysis occurs
at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated
2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis
of this unique metallocofactor is accomplished by three maturase enzymes
denoted HydE, HydF, and HydG. HydE and HydG belong to the radical <i>S</i>-adenosylmethionine superfamily of enzymes and synthesize
the nonprotein ligands of the H-cluster. These enzymes interact with
HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe
subcluster assembly. Prior characterization of HydF demonstrated the
protein exists in both dimeric and tetrameric states and coordinates
both [4Fe-4S]<sup>2+/+</sup> and [2Fe-2S]<sup>2+/+</sup> clusters
[Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick,
J. B. (2016) <i>Biochemistry 55</i>, 3514â3527].
Herein, electron paramagnetic resonance (EPR) is utilized to characterize
the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters bound to
HydF. Examination of spin relaxation times using pulsed EPR in HydF
samples exhibiting both [4Fe-4S]<sup>+</sup> and [2Fe-2S]<sup>+</sup> cluster EPR signals supports a model in which the two cluster types
either are bound to widely separated sites on HydF or are not simultaneously
bound to a single HydF species. Gel filtration chromatographic analyses
of HydF spectroscopic samples strongly suggest the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters are coordinated to the dimeric
form of the protein. Lastly, we examined the 2Fe subcluster-loaded
form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase
activation. Together, the results indicate a specific role for the
HydF dimer in the H-cluster biosynthesis pathway
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