Mechanistic studies of the radical S-adenosyl-l-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-d-quinovose

Article abstract of DOI:10.1021/ja201212f

DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to TDP-3-keto-4,6-dideoxy-d-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-d-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism. This hypothesis is now confirmed by EPR spectroscopy with the detection of a TDP-d-quinovose radical intermediate having a g-value of 2.0025 with hyperfine coupling to two spin 1/2 nuclei, each with a splitting constant of 33.6 G. A significant decrease in the EPR line width is observed when the radical is generated in reactions conducted in D ₂O versus H₂O. These results are consistent with a C3 β-hydroxyalkyl radical in which the p-orbital harboring the unpaired electron spin at C3 is periplanar with the C-H bonds at both C2 and C4.

Full text of DOI:10.1021/ja201212f

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Mechanistic Studies of the Radical S-Adenosyl-L-methionine Enzyme
DesII: EPR Characterization of a Radical Intermediate Generated
During Its Catalyzed Dehydrogenation of TDP-D-Quinovose
Mark W. Ruszczycky, Sei-hyun Choi, Steven O. Mansoorabadi, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin,
Austin, Texas 78712, United States
S Supporting Information
b
Scheme 1. Deamination and Dehydrogenation Reactions of
DesII
ABSTRACT: DesII, a radical S-adenosyl-L-methionine
(SAM) enzyme from Streptomyces venezuelae, catalyzes the
deamination of TDP-4-amino-4,6-dideoxy-D-glucose to
TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine bio-
synthetic pathway. DesII can also catalyze the dehydrogena-
tion of TDP-D-quinovose to the corresponding 3-keto sugar.
Similar to other radical SAM enzymes, DesII catalysis has
been proposed to proceed via a radical mechanism. This
hypothesis is now confirmed by EPR spectroscopy with the
detection of a TDP-D-quinovose radical intermediate having
a g-value of 2.0025 with hyperfine coupling to two spin 1/2
nuclei, each with a splitting constant of 33.6 G. A significant
decrease in the EPR line width is observed when the radical
is generated in reactions conducted in D2O versus H2O.
These results are consistent with a C3 R-hydroxyalkyl
radical in which the p-orbital harboring the unpaired elec-
tron spin at C3 is periplanar with the CꢀH bonds at both C2
and C4.
(1) to TDP-3-keto-4,6-dideoxy-D-glucose (2) (see Scheme 1).11,12
This enzyme has been characterized as a radical SAM enzyme on the
basis of the presence of a CxxxCxxC motif in its sequence, the
requirement of a reduced [4Feꢀ4S]1þ cluster and SAM for its
activity, and the transfer of a hydrogen atom from the C3 position of
1 to C50 of 50-deoxyadenosine during catalysis.13 Interestingly, when
the enzyme is incubated with TDP-D-quinovose (4) instead of the
natural substrate (1), analogous elimination of the C4 hydroxyl is
not observed; rather, the C3 hydroxyl is oxidized to the correspond-
ing ketone (5) with electron transfer back to the oxidized
[4Feꢀ4S]2þ cluster to regenerate the reduced [4Feꢀ4S]1þ
state.13,14 This chemistry resembles the dehydrogenation of
2-deoxy-scyllo-inosamine (6) to 3-amino-2,3-dideoxy-scyllo-inosose
(7) catalyzed by BtrN.15 While DesII is an established member of
the radical SAM enzyme family, experimental verification of the
expected radical intermediates during turnover has yet to be
provided. In this communication, we report the detection and
characterization of a substrate radical intermediate formed in the
DesII-catalyzed dehydrogenation of TDP-D-quinovose (4) to 5 by
electron paramagnetic resonance (EPR) spectroscopy.
he radical S-adenosyl-L-methionine (SAM) enzymes are an
T
important class of biocatalysts that are widespread in Nature.
While their catalytic functions are remarkably diverse, a reductive
homolytic cleavage of the C50ꢀS bond of SAM facilitated by a
[4Feꢀ4S]1þ cluster in the active site is a common step en route
to products for these enzymes.1ꢀ3 The resulting 50-deoxyadeno-
syl radical is used to abstract a hydrogen atom, commonly from
an unactivated CꢀH moiety, of the substrate in a manner
reminiscent of B12-dependent enzymes4,5 to initiate the chemical
reactions. The substrate radical intermediates so produced then
undergo further transformations distinctive for each enzyme to
generate the final products. Characterization of the proposed
substrate-derived radical intermediates is critical for functional
verification and may also lead to useful mechanistic insight
regarding the target enzyme. However, direct detection of these
radical intermediates is experimentally challenging due to the
transient nature of the radical species. Thus far, only a few radical
SAM enzymes for which successful detection of the substrate
radical intermediates has occurred have been reported. These
include lysine-2,3-aminomutase, HemN, and BtrN.6ꢀ9
When DesII is incubated with 4 (3:1 substrate/enzyme,
manually freeze-quenched at ∼11 s) an EPR signal is observed
In our study of the biosynthesis of TDP-desosamine (3)10 in
Streptomyces venezuelae we have identified a key enzyme, DesII, that
catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose
Received: February 8, 2011
Published: April 22, 2011
r
2011 American Chemical Society
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Figure 2. X-band EPR spectra of DesII (90 μM) in the presence of
250 μM TDP-D-[4-2H]quinovose (4D), 560 μM SAM, and 700 μM
Na2S2O4 in H2O and D2O (25 mM EPPS, 1 mM DTT, H2O pH 8.0).
Reactions were frozen using precooled isopentane approximately 11 s
after mixing. Overlayed red broken lines denote simulated spectra.
EPR acquisition parameters: microwave frequency, 9.45 GHz (H2O),
9.44 GHz (D2O); microwave power, 2.0 mW; receiver modulation
frequency, 100 kHz; receiver modulation amplitude, 5.0 G; signal
channel time constant, 5.12 ms; sweep time, 121 s; number of scans,
32 (H2O), 16 (D2O).
Figure 1. X-band EPR spectra of DesII (90 μM) in the presence of
250 μM TDP-D-quinovose (4), 560 μM SAM, and 700 μM Na2S2O4 in
H2O and D2O (25 mM EPPS, 1 mM DTT, H2O pH 8.0). Reactions
were frozen using precooled isopentane approximately 11 s after mixing.
Overlayed red broken lines denote simulated spectra. EPR acquisition
parameters: microwave frequency, 9.46 GHz (H2O), 9.44 GHz (D2O);
microwave power, 2.0 mW; receiver modulation frequency, 100 kHz;
receiver modulation amplitude, 5.0 G; signal channel time constant, 5.12
ms; sweep time, 121 s; number of scans, 32 (H2O), 16 (D2O).
as shown in Figure 1 (in H2O). The EPR spectrum can be well
simulated using an isotropic g-factor of 2.0025 with the inclusion
of isotropic hyperfine interactions with two equivalent spin 1/2
nuclei having splitting constants of 33.6 G (red broken line in
Figure 1). In line with previous experiments demonstrating
abstraction of a hydrogen atom from C3 of 1 during turnover,13
this doublet of doublets splitting pattern is consistent with a C3
radical of 4 with hyperfine couplings to the hydrogen atoms at C2
and C4. To validate this assignment, the C4-deuterated isoto-
pologue, TDP-D-[4-2H]quinovose (4D), was prepared by treat-
ment of TDP-4-keto-6-deoxy-D-glucose with NaBD4, followed
by HPLC separation from the minor epimeric byproduct, TDP-
D-[4-2H]fucose. As shown in Figure 2 (in H2O), when 4D is
reacted with DesII, the EPR signal collapses from a doublet of
doublets to a broadened doublet. EPR line shape simulations
utilizing the same g-factor (2.0025) and line width (7.2 G)
revealed that the doublet is due to coupling to a spin 1/2 nucleus
with a splitting constant of 33.6 G, whereas the broadening arises
from hyperfine coupling to a spin 1 nucleus with a splitting
constant of 5.2 G (red broken line in Figure 2). Such a reduction
in the magnitude of the splitting constant is consistent with that
expected from the ratio of the magnetogyric ratios (gH/gD),16
which is equal to the observed value of 6.5.
been subtracted from the spectra in Figures 1 and 2. Unfortu-
nately, attempts to capture a radical intermediate in the deamina-
tion of 1 by DesII were unsuccessful even when the reaction was
freeze-quenched within 100 ms of mixing enzyme with substrate.
The inability to trap such a radical during steady state turnover
may be related to the different redox reactions comprising the
catalytic cycles associated with the deamination of 1 versus the
dehydrogenation of 4.14
The above observation confirms the formation of a substrate-
derived radical intermediate in the DesII-catalyzed dehydrogena-
tion reaction. Judging from the equivalence of the hyperfine
splitting constants due to the C2 and C4 H-nuclei, the π-system
of the C3 centered radical is expected to have an equivalent
orientation with respect to the CꢀH bonds at C2 and C4. The
corresponding dihedral angle, χ, can be estimated from the
HellerꢀMcConnell relation,17
aðχÞ ¼ FðA1 þ A2 cos2 χÞ
with empirical values of 0.92 and 42.6 G for A1 and A2,
respectively.18 The Mulliken spin density, F, at C3 was computed
to be 0.83 by density functional theory (DFT) calculations on the
corresponding D-quinovose radical (see Supporting In-
formation). On the basis of the observed splitting constants of
33.6 G each, the dihedral angle, χ, between the p-orbital at the
sp2-hybridized C3 center and the adjacent C2ꢀH and C4ꢀH
Control experiments where either substrate or enzyme was
excluded showed only a small, nondescript EPR signal due to the
cavity (see Supporting Information). This background signal has
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Scheme 2. Proposed Mechanism for Dehydrogenation by
DesII
∼5 unit decrease in pKa of the C3 hydroxyl group to form the ketyl
radical 9 (Scheme 2).19 For the dehydrogenation of 4, deprotonation
of the hydroxyl group may drop the redox potential of the resulting
ketyl radical19 to a value sufficiently negative to facilitate electron
transfer from 9 back to the [4Feꢀ4S]2þ cluster to complete the
reaction. The shift of the reaction flux to electron transfer instead of
C4 elimination, as might be expected considering the deamination of
1 and the precedence set by the dioldehydratases,5 may in part be
explained by the observed geometry that places the C4 hydrogen, as
opposed to the C4 hydroxyl group, periplanar to the singly filled p-
orbital of the adjacent R-hydroxyalkyl radical. Consequently, the
different catalytic outcome of deamination in the case of 1 may result
from some combination of differences in nucleofugality of ammonia
versus hydroxide and a potentially different binding conformation
that is more conducive to the elimination of ammonia from C4.
In summary, the intermediacy of a C3 radical in the DesII-
catalyzed dehydrogenation of 4 has been firmly established by
EPR spectroscopy. This finding, in conjunction with previous
results, confirms that DesII catalysis follows radical SAM chem-
istry. The characteristics of the radical are consistent with an
R-hydroxyalkyl radical at the sp2-hybridized C3 position, where
the p-orbital harboring the unpaired electron spin is periplanar with
the CꢀH bonds at both C2 and C4. This conformation would
disfavor elimination of hydroxide at C4, while subsequent deproto-
nation to a C3 ketyl radical is expected to promote electron transfer
to the [4Feꢀ4S]2þ cluster. Though similar chemistry is observed
with BtrN, for which dehydrogenation is its primary role in vivo,
BtrN has recently been shown to utilize a second [4Feꢀ4S] cluster
to effect the electron transfer.20 In contrast, DesII (having only four
cysteine residues) only possesses one [4Feꢀ4S] complex,13 such
that dehydrogenation of TDP-quinovose may proceed via direct
electron transfer back to the oxidized cluster. How this might relate
to deamination and the relative timing of electron transfer versus
deprotonation during dehydrogenation remains unanswered and is
currently being pursued in the study of this interesting example of
radical SAM chemistry.
bonds is thus predicted to be ∼15.0°. Were all the spin density to
be localized at C3, i.e., F = 1, the dihedral angle would remain no
greater than 30°. These observations are consistent with a chair
conformation of the TDP-D-quinovose radical intermediate in
which the p-orbital at C3 is nearly eclipsed by the CꢀH bonds at
the two adjacent carbon centers. A comparable geometry has also
been reported for the BtrN substrate radical intermediate.8
To gain more information about the ionization state of the TDP-
D-quinovose radical, enzymatic incubations in D2O were also carried
out. The corresponding EPR spectra of the reaction mixtures are
presented in Figures 1 (D2O) and 2 (D2O) along with results from
the simulation of the EPR line shapes (red broken lines). The
radicals derived from either isotopologue (4 and 4D) of TDP-
D-quinovose exhibit a marked narrowing of the line widths of their
EPR signals from 7.2 to 5.0 G when H2O is exchanged for D2O. In
the case of 4D, this narrowing permits clear visualization of the
hyperfine splitting of the EPR signal due to the C4 deuterium
nucleus. The narrowing of the line widths strongly suggests that the
C3 hydroxyl group of the observed radical is not deprotonated.
Thus, by replacing •C3ꢀOH with •C3ꢀOD, the larger splitting
from H is reduced to that of D leading to an apparent reduction in
inhomogenous broadening of the EPR signal. Interestingly, DFT
calculations on the D-quinovose ketyl radical, obtained by deproto-
nation of the C3 hydroxyl group, yields a F value of 0.63. This value
is inconsistent with the large magnitude of the experimental
hyperfine splitting constants and, thus, supports the assigned ioniza-
tion state of the •C3ꢀOH radical.
’ ASSOCIATED CONTENT
S
Supporting Information. Details regarding experimental
b
procedures, NMR characterization of TDP-D-[4-2H]quinovose,
EPR line shape simulation, and DFT calculations as well as a list
of abbreviations. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
’ ACKNOWLEDGMENT
This work was supported in part by grants from the National
Institutes of Health (GM35906 and GM54346) and a fellowship
award (F32AI082906) from the National Institute of Allergy and
Infectious Diseases (to M.W.R.).
Although, the radical detected in these experiments is an R-
hydroxyalkyl radical (•C3ꢀOH), deprotonation of the hydroxyl
group to form the corresponding ketyl radical (•C3ꢀOꢀ) is
believed to be important for DesII catalysis. One hypothesis
posits that the C3 radical (8), after formation, can readily be
deprotonated by an active site base, facilitated by an expected
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