Mechanistic studies of the radical S-adenosylmethionine enzyme DesII with TDP-D-fucose

Article abstract of DOI:10.1002/anie.201409540

DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDP-quinovose (TDP-fucose) was examined. The reaction primarily results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation.

Full text of DOI:10.1002/anie.201409540

Angewandte
Chemie
DOI: 10.1002/anie.201409540
Biosynthesis
Mechanistic Studies of the Radical S-Adenosylmethionine Enzyme
DesII with TDP-d-Fucose**
Yeonjin Ko, Mark W. Ruszczycky, Sei-Hyun Choi, and Hung-wen Liu*
Abstract: DesII is a radical S-adenosylmethionine (SAM)
enzyme that catalyzes the C4-deamination of TDP-4-amino-
4,6-dideoxyglucose through a C3 radical intermediate. How-
ever, if the C4 amino group is replaced with a hydroxy group
(to give TDP-quinovose), the hydroxy group at C3 is oxidized
to a ketone with no C4-dehydration. It is hypothesized that
many macrolide antibiotics.[2–4] This deamination reaction is
radical-mediated and is initiated by hydrogen atom abstrac-
tion from the substrate by a 5’-deoxyadenosyl radical. The
latter is derived from the reductive homolysis of SAM by an
active site [4Fe-4S]1+ cluster and represents the hallmark of
radical SAM enzymology.[5]
ꢀ
hyperconjugation between the C4 C N/O bond and the
Two general mechanisms have been proposed for DesII-
catalyzed deamination (Figure 1).[1,4] In both cases, the
p orbital harboring the unpaired electron at C3 of the radical
partially filled p orbital at C3 of the radical intermediate
modulates the degree to which elimination competes with
dehydrogenation. To investigate this hypothesis, the reaction of
DesII with the C4-epimer of TDP-quinovose (TDP-fucose)
was examined. The reaction primarily results in the formation
of TDP-6-deoxygulose and likely regeneration of TDP-fucose.
The remainder of the substrate radical partitions roughly
equally between C3-dehydrogenation and C4-dehydration.
Thus, changing the stereochemistry at C4 permits a more
balanced competition between elimination and dehydrogen-
ation.
T
he radical S-adenosylmethionine (SAM) enzyme DesII
from Streptomyces venezuelae catalyzes the redox-neutral
deamination of TDP-4-amino-4,6-dideoxy-d-glucose (1) to
generate TDP-4,6-dideoxy-3-keto-d-glucose (2, TDP = thy-
midine diphosphate; Scheme 1).[1,2] In its biological context,
the deamination of 1 is the key reaction in the biosynthesis of
TDP-desosamine (3), which is an essential component of
Figure 1. Possible reaction pathways for the substrate radical inter-
mediate 6 during the DesII catalytic cycle. When R=NH3+, 6 may
undergo either an elimination (e.g., 6!8!9) or 1,2-migration (6!
7!9/10) to produce 2. When R=OH, 6 undergoes an oxidation, likely
via the ketyl radical intermediate 8 to produce 5.
ꢀ
intermediate 6 must overlap productively with the C N s-
system at C4 in order to facilitate either 1,2-migration (6!
7!9/10!2) or direct elimination (6!8!9!2) of the
adjacent amino group. In this regard, DesII is highly
reminiscent of ethanolamine ammonia lyase (EAL), which
catalyzes the deamination of ethanolamine, albeit using a 5’-
deoxyadenosyl radical produced from the homolysis of
adenosylcobalamin rather than SAM.[6] Although the chemis-
try of DesII is also very similar to the dehydration of 1,2-diols
by the B12-dependent dioldehydratases,[7] no elimination of
the C2 hydroxyl from 1 is observed during the catalytic cycle
of DesII.
Scheme 1. Reactions catalyzed by DesII.
[*] Y. Ko, M. W. Ruszczycky, S.-H. Choi,[+] Prof. Dr. H.-w. Liu
Division of Medicinal Chemistry, College of Pharmacy
and Department of Chemistry, University of Texas at Austin
Austin, TX 78712 (USA)
E-mail: h.w.liu@mail.utexas.edu
[+] Current address: Department of Chemistry
DesII can also accept TDP-d-quinovose (4), in which the
C4 amino group of 1 is replaced with a hydroxy group, as
a substrate. However, DesII does not catalyze elimination of
the C4 hydroxy group from 4 to produce 2, but rather
catalyzes oxidation of the C3 hydroxy group to yield 5
(Scheme 1).[1] This second, dehydrogenase activity of DesII is
The Scripps Research Institute
San Diego, CA 92037 (USA)
[**] This work was supported by grants from the National Institutes of
Health (GM035906) and the Welch Foundation (F-1511).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201409540.
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analogous to the oxidation of 3-deoxy-scyllo-inosamine by the
radical SAM enzyme BtrN from the butirosin biosynthetic
pathway[8] and the dehydrogenation of a cysteine or serine
residue to formylglycine catalyzed by anaerobic sulfatase
maturating enzymes.[9] The dual capability of DesII to operate
as a lyase in one instance and a dehydrogenase in another
offers an ideal model system for investigating the subtleties of
radical control within an enzyme active site.
Previous EPR investigation of the dehydrogenation
reaction (4!5) catalyzed by DesII identified a C3 a-
ꢀ
hydroxyalkyl radical intermediate in which the C O bonds
at both C2 and C4 are essentially orthogonal to the p orbital
at C3.[10] While the structure of the radical intermediate
during deamination has yet to be described, these observa-
tions led to the hypothesis that a difference in the binding
configuration of the substrate radical is important for
determining the partitioning of the C3-centered radical
intermediate between the elimination and oxidation path-
ways (i.e., 6!2 versus 6!5). It is proposed that whenever the
configuration at C4 provides sufficient hyperconjugation
Figure 2. HPLC traces showing the consumption of TDP-d-fucose (12)
and SAM in the presence of DesII and Na2S2O4 at pH 8.0. Trace 1 was
measured after 2 h without DesII. Traces 2 and 3 were measured after
30 min and 6 h in the presence of DesII, respectively. The retention
times in traces 2 and 3 are shifted by 2 and 4 min compared to
trace 1. Three new products (X, Y, and Z) are observed as shown in
the inset (from Trace 3). The formation of 5’-deoxyadenosine (5’-dAdo)
is also noted. Peak A corresponds to the methylthioadenosine decom-
position product of SAM.[14] Peak B corresponds to thymidine mono-
phosphate. Peak C indicates a contaminant in the SAM reagent.
ꢀ
between the C4 C N/O s system and the C3 p orbital, then
elimination proceeds more rapidly than electron transfer to
the [4Fe-4S]2+ cluster, and lyase activity is observed. By
contrast, if the configuration does not provide good overlap,
then elimination is impeded. In this case, the more strongly
reducing ketyl radical (8b) leads to faster reduction of the
cluster compared to elimination of the C4 moiety, and
dehydrogenase activity is observed.[2,11] Such a mechanism
would help to explain why the C2 (in 1 and 4) and C4 (in 4)
hydroxy groups are inert to dehydration during DesII
catalysis, while a,b-dihydroxyalkyl radicals generated in
solution through pulse radiolysis undergo rapid dehydra-
tion.[12] This working model implies that configurational
inversion at C4 of the TDP-d-quinovose substrate radical
(6, R = OH) could result in C4 dehydratase activity if such
a stereochemical change permitted better hyperconjugation.
This proposal can be tested by employing the C4 epimer of 4
(TDP-d-fucose (12); Scheme 3) as a potential substrate for
DesII. Reported herein are the results and mechanistic
implications of our studies of DesII using 12 as the substrate.
TDP-d-fucose (12) was prepared from TDP-d-glucose
with 4,6-dehydratase RfbB followed by reduction with
NaBH4 as previously described (see the Supporting Informa-
tion).[1,13] The consumption of both 12 and SAM was only
observed upon prolonged incubation (hours) with a high
concentration (10–20 mm) of DesII in the presence of
Na2S2O4. This corresponded to a specific activity of approx-
imately 5 ꢀ 10ꢀ4 mmolminꢀ1 mgꢀ1 for the consumption of 12
versus 1 mmolminꢀ1 mgꢀ1 for the dehydrogenation of 4 at
pH 8.0.[11] The much lower specific activity of DesII towards
12 helps to explain why it has not previously been recognized
as a substrate.[1]
adduct of deoxyadenosine (neutral mass: 315.1 Da). This
hypothesis was supported by the observation of ESI-MS
peaks at m/z 318.1 (positive ion Xd; Figure 3) and m/z 316.1
(negative ion) when [5’,5’-2H2]SAM was used instead of SAM.
Further investigation showed that species X was also gener-
ated during prolonged co-incubation of DesII, SAM, and
Na2S2O4 in the absence of 12; however, it was not formed if
either SAM, DesII, or Na2S2O4 was excluded from the
Three new products, in addition to 5’-deoxyadenosine,
were observed by HPLC in the reaction of DesII with 12
(Figure 2). The product peak X with a retention time of
28.4 min was isolated and characterized by electrospray
ionization mass spectrometry (ESI-MS). The detection of
peaks at m/z 316.1 (positive ion X; Figure 3) and m/z 314.1
(negative ion) suggested that species X may be a sulfinate
Figure 3. ESI-MS of isolated HPLC peaks X (positive ion), Y (negative
ion), and Z (negative ion) from Figure 2. The spectrum in Xd (positive
ion) corresponds to species X isolated from the reaction by using
[5’,5’-2H2]SAM. The peak at m/z 338.3 likely corresponds to a known
contaminant (i.e., erucamide) in the ESI-MS rather than the [M+Na]+
peak of X.
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Scheme 2. Conversion of SAM to 11 in the presence of DesII and
Na2S2O4
reaction mixture. Based on these observations, species X was
assigned as 5’-deoxyadenosyl-5’-sulfinic acid (11; Scheme 2).
The formation of sulfinate adducts has been reported in
the reactions of two other radical SAM enzymes when
Na2S2O4 is used to maintain a reduced [4Fe-4S]1+ cluster. For
example, a mutant of spore photoproduct lyase has been
shown to catalyze the production of a sulfinic acid adduct of
dithymidine monophosphate.[15] The atypical radical SAM
enzyme Dph2, which employs a SAM-derived 3-amino-3-
carboxypropyl (ACP) radical rather than a 5’-deoxyadenosyl
radical, has also been shown to produce a sulfinic acid
derivative of ACP during turnover.[16] However, to our
knowledge, the conversion of SAM into 11 in the presence
of DesII and Na2S2O4 is the first report of a sulfinate
derivative of 5’-deoxyadenosine being generated by a radical
SAM enzyme. The specific activity for the sulfination reaction
is no greater than 2 ꢀ 10ꢀ3 mmolminꢀ1 mgꢀ1 in the absence of
TDP-d-fucose and its rate of formation is reduced in the
presence of the TDP-sugar. This result suggests that dithionite
can access the DesII active site and intercept the 5’-
deoxyadenosyl radical, especially in the absence of a sugar
substrate.
Both of the remaining two product peaks in Figure 2
originated from TDP-d-fucose (12). ESI-MS analysis of the
major product peak (Y, retention time 30.5 min) showed
signals indicative of [MꢀH]ꢀ and [Mꢀ2H+Na]ꢀ ions at m/z
547.1 and 569.1, respectively (Figure 3). This result is
consistent with an isomer of TDP-d-fucose; however, the
HPLC retention time and relative inertness to reaction with
DesII ruled out assignment as TDP-d-quinovose (4). Spe-
cies Y was found to be sufficiently stable to permit collection
for 1H NMR analysis, and all coupling constants between
protons on the hexose ring are relatively small (< 6 Hz), thus
Scheme 3. Summary of reactions catalyzed by DesII when TDP-d-
fucose (12) serves as the substrate.
product 15. Furthermore, 15 went from being the major
distinguishable product (ca. 80%) in H2O to a minor product
(< 30%) in D2O, thus indicating a solvent deuterium kinetic
isotope effect on the partitioning of 13 between the different
routes of decomposition. The deuterium content of the
residual TDP-d-fucose in the D2O buffers was also inves-
tigated by mass spectrometry. Small but measurable increases
in the deuteration of the residual substrate (12) were
observed, thus suggesting that net H-atom transfer is possible
to both faces of the C3 radical. These results indicate that 13 is
solvent accessible, and the H atom transferred to C3 origi-
nates from a solvent-exchangeable source (see the Supporting
Information).
The later eluting peak Z from the DesII reaction with
TDP-d-fucose at 34.3 min co-eluted with the deamination
product 2. Furthermore, negative mode ESI-MS of the
collected peak exhibited a signal at m/z 529.1 (Figure 3).
This value is consistent with its assignment as the dehydration
product 2. To verify the identity of species Z as 2, the DesII
reaction with 12 was further treated with the transaminase
DesV, which catalyzes the reductive amination of 2 in the
presence of glutamate.[17,18] This resulted in the disappearance
of peak Z and formation of a new HPLC peak that co-eluted
with TDP-3-amino-3,4,6-trideoxy-d-glucose (16) as predicted
(see the Supporting Information). These observations indi-
cate that DesII is indeed capable of operating as a dehydra-
tase.
ꢀ
indicating an absence of trans-diaxial C H bonds. This result
implies a diaxial configuration of the vicinal hydroxyl groups
at C3 and C4 and led to the assignment of species Yas TDP-6-
deoxy-d-gulose (15), which is the C3-epimer of TDP-d-
fucose. However, the 1H NMR spectra of species Y exhibited
significant contamination due to partial decomposition, and
a standard of 15 was prepared in order to confirm the
1
assignment by both HPLC coinjection and H NMR spec-
The dehydratase activity of DesII was also investigated by
using a DesII/DesV coupled reaction system. Under these
conditions, however, TDP-3-amino-3,4,6-trideoxy-d-glucose
(16) was formed in a roughly 1:1 ratio with an additional peak
at a retention time also consistent with a TDP-aminosugar.
Negative ion ESI-MS analysis of the new peak revealed
a signal at m/z 546.1, thus suggesting the [MꢀH]ꢀ ion of TDP-
3-amino-3-deoxy-d-fucose (17). To confirm this assignment,
the 3,4-ketoisomerase FdtA from Aneurinibacillus thermoaer-
troscopy (see the Supporting Information). The formation of
TDP-6-deoxy-d-gulose from TDP-d-fucose implies that net
H-atom return to the C3 radical intermediate (13; Scheme 3)
of TDP-d-fucose is also possible in addition to dehydration
and dehydrogenation (see below).
When the reaction was run in buffer containing at least
95% deuterium, ESI-MS analysis showed an approximately
four-fold incorporation of deuterium versus protium into the
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ophilus was used in combination with DesV and TDP-6-
deoxy-4-keto-d-glucose to produce a standard of TDP-3-
amino-3-deoxy-d-fucose (17).[19,20] This standard indeed co-
eluted by HPLC with the second DesII/DesV product from
the TDP-d-fucose reaction (see the Supporting Information).
The fact that the dehydrogenation product TDP-3-keto-d-
fucose (14) was not directly observed during the reaction of
TDP-d-fucose with DesII alone may be attributed to the poor
stability of 3-keto TDP-sugars, which tend to readily decom-
pose to TDP and ketodihydropyrans.[21] The observation that
both TDP-3-amino-3,4,6-trideoxy-d-glucose (16) and TDP-3-
amino-3-deoxy-d-fucose (17) are generated in an approx-
imately 1:1 ratio in the DesII/DesV reaction indicates that
dehydration and dehydrogenation of TDP-d-fucose (12)
compete with one another to an approximately equal extent
and without racemization at C4.
In summary, despite being a poor substrate, TDP-d-fucose
(12) can be recognized by DesII, whereupon it undergoes H-
atom abstraction at C3 (12!13; Scheme 3). The fate of the
resulting C3-centered radical intermediate 13 is of interest
because of its inverted stereochemistry at C4 compared to
TDP-d-quinovose (4). The majority of 13 is reduced to
produce TDP-6-deoxy-d-gulose (13!15) through net H-
atom transfer from a solvent-exchangeable source, and
regeneration of TDP-d-fucose (13!12) likely also takes
place through an analogous process. Therefore, the altered
geometry at C4 of 12 compared to 1 and 4 appears to
destabilize the enzyme–intermediate complex, thereby per-
mitting access to the radical by the solvent or leading to the
dissociation of the complex.
Keywords: biosynthesis · carbohydrates · enzyme catalysis ·
radical reactions · S-adenosylmethionine
.
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not undergo reduction to 15/12, approximately 50% is
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+
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ꢀ
C N bond cleavage during the deamination of 1.
Received: September 26, 2014
Published online: && &&, &&&&
4
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Biosynthesis
What dictates the substrate’s fate? DesII
is a radical S-adenosylmethionine (SAM)
enzyme that can catalyze either deamin-
ation or dehydrogenation depending on
the substitution pattern of its substrate.
By altering the stereochemistry of the
dehydrogenation substrate, however,
dehydration also becomes possible. This
supports a model in which the fate of the
substrate radical depends on its binding
configuration in the enzyme active site.
Y. Ko, M. W. Ruszczycky, S.-H. Choi,
H.-w. Liu*
&&&& — &&&&
Mechanistic Studies of the Radical S-
Adenosylmethionine Enzyme DesII with
TDP-d-Fucose
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