Radical SAM Activation of the B12-Independent Glycerol Dehydratase Results in Formation of 5′-Deoxy-5′-(methylthio) adenosine and Not 5′-Deoxyadenosine

Article abstract of DOI:10.1021/bi101255e

Activation of glycyl radical enzymes (GREs) by S-adenosylmethonine (AdoMet or SAM)-dependent enzymes has long been shown to proceed via the reductive cleavage of SAM. The AdoMet-dependent (or radical SAM) enzymes catalyze this reaction by using a [4Fe-4S] cluster to reductively cleave AdoMet to form a transient 5′deoxyadenosyl radical and methionine. This radical is then transferred to the GRE, and methionine and 5′deoxyadenosine are also formed. In contrast to this paradigm, we demonstrate that generation of a glycyl radical on the B12-independent glycerol dehydratase by the glycerol dehydratase activating enzyme results in formation of 5′deoxy- 5′(methylthio) adenosine and not 5′deoxyadenosine. This demonstrates for the first time that radical SAM activases are also capable of an alternative cleavage pathway for SAM.

Full text of DOI:10.1021/bi101255e

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40 Biochemistry 2011, 50, 440–442
DOI: 10.1021/bi101255e
Radical SAM Activation of the B -Independent Glycerol Dehydratase Results in
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Formation of 5 -Deoxy-5 -(methylthio)adenosine and Not 5 -Deoxyadenosine
Jonathan M. Demick and William N. Lanzilotta*
Department of Biochemistry and Molecular Biology, A220B Davison Life Sciences Building, University of Georgia,
Athens, Georgia 30602, United States
Received August 5, 2010; Revised Manuscript Received December 23, 2010
ABSTRACT: Activation of glycyl radical enzymes (GREs)
by S-adenosylmethonine (AdoMet or SAM)-dependent
enzymes has long been shown to proceed via the reductive
cleavage of SAM. The AdoMet-dependent (or radical
SAM) enzymes catalyze this reaction by using a [4Fe-4S]
cluster to reductively cleave AdoMet to form a transient
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5
-deoxyadenosyl radical and methionine. This radical is then
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transferred to the GRE, and methionine and 5 -deoxyade-
nosine are also formed. In contrast to this paradigm, we
demonstrate that generation of a glycyl radical on the
B -independent glycerol dehydratase by the glycerol dehydra-
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tase activating enzyme results in formation of 5 -deoxy-
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5
-(methylthio)adenosine and not 5 -deoxyadenosine. This
demonstrates for the first time that radical SAM activases
are also capable of an alternative cleavage pathway for
SAM.
The identification of the “radical SAM” or “AdoMet radical”
superfamily has fundamentally changed our view of the utility
and functional diversity of the cofactor S-adenosylmethionine
(
“
AdoMet or SAM) (1-4). This report is focused on radical SAM
activases” involved in the generation of a catalytic glycyl radical
on glycyl radical enzymes (GREs). Pyruvate formate lyase
activating enzyme (PFL-AE) was one of the first radical SAM
activases to be characterized and performs the activation of the
GRE, pyruvate formate lyase (PFL) (5-18). The mechanistic
paradigm that has been established for generation of the glycyl
radical on essentially all GREs is shown in Figure 1A. Our group
has previously reported the isolation of a GRE capable of
dehydrating glycerol (19, 20). Similar to PFL, the glycerol
dehydratase (GD) was activated by another enzyme termed the
glycerol dehydratase activating enzyme (GD-AE) and required
AdoMet and strictly anoxic conditions (19). In this work, we have
monitored the activation of the GD by the GD-AE and show
that, as for several other GREs, a glycyl radical is formed. How-
ever, in contrast to the current paradigm, formation of the glycyl
F
IGURE 1: Proposed cleavage pathways during glycyl radical gen-
eration as catalyzed by radical SAM activases. The mechanism
observed for the pyruvate formate lyase (A) and an alternative
pathway proposed for the B12-independent glycerol dehydratase
(
B) are depicted.
AdoMet. AdoMet also appears to perturb the g values of the
Fe-S cluster signal (in Figure 2, compare panel A with panel B).
Upon addition of the GD to the mixture of AdoMet and the
GD-AE, containing a reduced Fe-S cluster, radical formation is
observed (Figure 2C). The g values for the observed radical are
identical to what has been observed for PFL and anaerobic
ribonucleotide reductase (ARNR) and not that surprising given
the high degree of sequence identity between these enzymes.
The complexity of the EPR signature for the GD-AE is
consistent with the presence of the additional [4Fe-4S] cluster
binding motifs in the primary structure of the GD-AE and
multiple Fe-S clusters that may or may not be spin-coupled.
The precise composition and electronic structure of the cluster or
clusters that give rise to the EPR spectra are currently under
investigation as well as the stoichiometry of the conversion of
cluster spin to radical spin observed during activation.
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radical results in formation of 5 -deoxy-5 -(methylthio)adenosine
MTA). These data suggest that some radical SAM activases may
(
use an alternative cleavage mechanism (Figure 1B) during acti-
vation of their GRE and suggest that a different radical inter-
mediate may exist for certain radical SAM activases.
Activation of the GD by the GD-AE as monitored by EPR
spectroscopy is shown in Figure 2. Formation of the glycyl
radical requires a reduced iron-sulfur cluster (Figure 2A) and
†
This work was also supported in part by a cooperative agreement with
Metabolic Explorer and Petroleum Research (44563-AC4 to W.N.L.).
While the formation of the glycyl radical was somewhat
anticipated, the observation that the activation mechanism
*To whom correspondence should be addressed. E-mail: wlanzilo@
bmb.uga.edu. Phone: (706) 542-1324. Fax: (706) 542-1738.
pubs.acs.org/biochemistry
Published on Web 12/23/2010
r 2010 American Chemical Society

Rapid Report
Biochemistry, Vol. 50, No. 4, 2011 441
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S-adenosylmethionine, 5 -deoxyadenosine, and 5 -deoxy-5 -(methyl-
IGURE 3: HPLC analysis of assay buffer containing a mixture of
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thio)adenosine (AdoMet, 5 -dAdo, MTA), only 5 -deoxy-5 -(met-
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hylthio)adenosine (MTA), only 5 -deoxyadenosine (5 -dAdo), or the
complete assay mix required for activation of the GD with (þ GD-
AE) or without (No GD-AE) the GD-AE. The asterisk highlights the
appearance of a peak in the HPLC chromatogram when the reaction
is quenched 15 s after the addition of the GD-AE.
MTA. By analogy, we propose that a radical intermediate,
similar to what has been reported for Dhp2, abstracts a hydrogen
atom to generate the glycyl radical on the GD. This is the only
way to account for formation of MTA and 2-aminobutyric acid.
While the precise identity of the radical intermediate in the
GD-AE activation mechanism is currently being investigated,
this is the first time that MTA and 2-aminobutyrate have been
observed as reaction products during the activation of a GRE by
a radical SAM activase. The data clearly demonstrate that
different reductive cleavage pathways exist for radical SAM
enzymes. Moreover, these results are extremely surprising given
the level of identity in the primary structures for PFL, ARNR,
and the GD. Whether radical SAM activating enzymes use the
alternative mode of AdoMet cleavage during radical generation
is now an important question that needs to be investigated
further. Given the structural similarities between the GD and
PFL at the level of both primary and tertiary structure, it is
difficult at this time to determine exactly why the GD-AE follows
a different reductive cleavage pathway during glycyl radical
formation. Ultimately, to address this question, some structural
information will be required.
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IGURE 2: EPR spectra of the GD-AE alone (A), in the presence of
mM SAM (B), and in the presence of 5 mM SAM as well as the GD
(C). EPR parameters and sample preparation are described in detail
in the Supporting Information.
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results in the formation of MTA and not 5 -deoxyadenosine is
exciting because every other radical SAM activase investigated to
date has been shown to produce methionine and 5 -deoxyade-
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nosine during generation of the glycyl radical (3, 21). Figure 3
shows the activation reaction as monitored by high-performance
liquid chromatography (HPLC). No MTA peak was observed if
either the GD or the GD-AE were excluded from the assay.
Calibration of the MTA peak area against a standard curve
revealed a stoichiometric relationship between the amount of GD
added and the amount of MTA formed. To detect any amino
acid products of activation, we applied Edman derivatization of
the reaction mixture and HPLC. Consistent with our proposal,
we observe simultaneous formation of 2-aminobutyrate and
MTA (Supporting Information). Regardless of the radical inter-
mediate that is formed as a result of reductive cleavage of
AdoMet, our results are significant because it has been widely
accepted that all radical SAM enzymes exclusively cleave the
C5 -S bond. Figure 3 and Figure S2 of the Supporting Informa-
tion show that, at least for the GD-AE, this is not the case. The
prevailing wisdom states that reductive cleavage of SAM should
result in a transient 5 -deoxyadenosyl radical that would rapidly
abstract a hydrogen atom from the conserved glycine residue on
the GD (2, 3, 22). In this case, the cleavage products are
methionine and 5 -deoxyadenosine. Regardless of the radical
PFL-AE, anerobic ribonucleotide reductase activating enzyme
(ARNR-AE), and the GD-AE all contain the hallmark adeno-
sine and catalytic cluster (CX CX C) binding motifs. However,
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in contrast to both PFL-AE and ARNR-AE, the GD-AE also
contains two additional ferredoxin-like [4Fe-4S] cluster binding
domains (20). The latter observation is also true for at least two
other radical SAM enzymes involved in GRE activation, speci-
fically, BssD and hydroxyphenylacetate decarboxylase acti-
vase (24, 25). At this time, the chemical products of glycyl
radical formation in the latter two systems have not been
investigated. The physiological role and exact composition of
the additional clusters in these enzymes have not been fully
investigated, but a reasonable hypothesis is that the additional
domain is required for the GD-AE to accept electrons from
a physiological donor, as proposed for hydroxyphenylacetate
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intermediate, which is not the focus of this work, there is
precedence for an alternative reductive cleavage event that leads
to MTA formation (23) as we have observed. Although it is not a
radical SAM enzyme, Dhp2 contains a [4Fe-4S] cluster that has
recently been shown to catalyze the reductive cleavage of
AdoMet (23) to form a 3-amino-3-carboxypropyl radical and

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42 Biochemistry, Vol. 50, No. 4, 2011
Demick and Lanzilotta
decarboxylase activase based on mutagenesis results (25). The
electron donor for the GD-AE is most likely a low-potential
ferredoxin. This is in contrast to PFL-AE for which it has been
shown that electrons for PFL-AE are provided by reduced
flavodoxin (8). While the source of the low-potential electron
required for reductive cleavage of AdoMet may seem like a minor
point, this may lead to a different structural arrangement in the
active site. Moreover, it is not known whether other AdoMet
radical activating enzymes containing the additional ferredoxin-
like [4Fe-4S] cluster binding domains also work by reductive
cleavage of AdoMet to yield MTA. In any case, our findings
provide evidence for an exciting new hypothesis, specifically
that, in contrast to the current paradigm, radical SAM activases
containing additional Fe-S clusters may operate by a different
reductive cleavage mechanism. At present, PFL-AE is the only
structure available for any radical SAM activase. A structure of a
radical SAM activase with the additional ferredoxin-like [4Fe-4S]
cluster binding domains will certainly provide some insight into
the atomic details of AdoMet binding, but additional parameters
such as the kinetics and thermodynamics of electron transfer and
the reductive cleavage event must also be investigated. Whether
one reductive cleavage mechanism is chosen over the other
and the occurrence of the additional Fe-S cluster-containing
domains in radical SAM activases throughout Nature are likely
to have important evolutionary implications. For example, one
might inquire about whether PFL-AE is truly a model enzyme for
all radical SAM activases or whether PFL-AE is actually an
exception to the rule.
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