Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme desll

Article abstract of DOI:10.1021/ja909451a

Desll from Streptomyces venezuelae is a radical SAM (S-adenosyl-L- methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D- glucose to form TDP-3-keto-4,6-dideoxy-D-glucose in the biosynthesis of TDP-D-desosamine. Desll also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-D-glucose. These properties prompted an investigation of how Desll handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-Dglucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, Desll is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 ± 0.05 and 1.01 ± 0.05 in the deamination and dehydrogenation reactions, respectively, using Na₂S₂O₄ as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP⁺ oxidoreductase/NADPH reducing system is used in place of Na₂S₂O₄. Moreover, there is no evidence of an uncoupled or abortive process in the deamination reaction, as indicated by the observation that dehydrogenation can take place in the absence of an external source of reductant whereas deamination cannot. Mechanistic and biochemical implications of these results are discussed.

Full text of DOI:10.1021/ja909451a

Published on Web 02/01/2010
Stoichiometry of the Redox Neutral Deamination and
Oxidative Dehydrogenation Reactions Catalyzed by the
Radical SAM Enzyme DesII
Mark W. Ruszczycky, Sei-hyun Choi, 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
Received November 10, 2009; E-mail: h.w.liu@mail.utexas.edu
Abstract: DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-L-methionine) enzyme that
catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to form TDP-3-keto-4,6-dideoxy-D-glucose
in the biosynthesis of TDP-D-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological
substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-D-glucose. These properties prompted an investigation
of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions.
This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-D-
glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate.
In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 ( 0.05 and
1.01 ( 0.05 in the deamination and dehydrogenation reactions, respectively, using Na2S2O4 as the reductant.
Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP+
oxidoreductase/NADPH reducing system is used in place of Na2S2O4. Moreover, there is no evidence of
an uncoupled or abortive process in the deamination reaction, as indicated by the observation that
dehydrogenation can take place in the absence of an external source of reductant whereas deamination
cannot. Mechanistic and biochemical implications of these results are discussed.
Scheme 1
Radical SAM enzymes are an important class of biological
catalysts involved in radical mediated transformations in both
primary and secondary metabolic pathways.1-3 All of these
enzymes contain a [4Fe-4S] cluster in the active-site and are
S-adenosyl-L-methionine (1, SAM) dependent. The reactions are
initiated by a single electron transfer from the reduced
[4Fe-4S]1+ cluster to SAM (1 f 2) to induce the homolytic
cleavage of the C5′-S bond in SAM, which yields a reactive
5′-deoxyadenosyl radical (4) along with L-methionine (3), as
shown in Scheme 1.2,4-6 Utilization of a 5′-deoxyadenosyl
radical (4) by the radical SAM enzymes is similar to the B12-
dependent enzymes. In both cases, the 5′-deoxyadenosyl radical
is used to generate a substrate radical intermediate that is
subsequently converted to product.1,2 However, unlike the
radical SAM enzymes, the 5′-deoxyadenosyl radical (4) in the
B12-dependent enzymes is derived from an adenosylcobalamin
cofactor.7-9 Moreover, the B12-dependent enzymes are primarily
involved in redox neutral isomerization and lyase reactions,
whereas the reactions catalyzed by radical SAM enzymes may
or may not lead to a net change in the redox state of the substrate
as it is converted to product. Thus, the fact that SAM can act
as both a radical initiator as well as an oxidant highlights a
clear difference between those enzymes utilizing adenosylco-
balamin versus radical SAM chemistry.
In the case of the adenosylcobalamin-dependent enzymes,
isomerization reactions are typically accompanied by the
regeneration of the adenosylcobalamin cofactor. Similarly, SAM,
acting as a coenzyme, is also regenerated in the redox neutral
transformations catalyzed by radical SAM dependent lysine 2,3-
aminomutase (LAM)5,10,11 and spore photoproduct lyase (SPL)
(see Scheme 2A).12,13 However, for the radical SAM enzymes
catalyzing transformations that lead to an overall oxidation of
the substrate, there is a net consumption of SAM. Examples
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A R T I C L E S
Ruszczycky et al.
Scheme 2
include the sulfur insertion reactions catalyzed by biotin synthase
(BS)14-16 and lipoyl synthase (LipA) (see Scheme 2B).17-19
These reactions involve four-electron oxidation of dethiobiotin
(5) to biotin (6) and octanoyl-LCP (7) to lipoyl-LCP (8), where
LCP denotes a lipoyl carrier protein. A net four-electron
oxidation/decarboxylation of coproporphyrinogen III (9) by
HemN has also been studied (Scheme 2B).20,21 The dehydro-
genation of 2-deoxy-scyllo-inosamine (10) to 3-amino-2,3-
dideoxy-scyllo-inosose (11) by BtrN,22 and the oxidative
desulfuration of cysteinyl (12) to formylglycyl residues (13) by
anaerobic sulfatase maturating enzymes (anSMEs) are examples
of two-electron oxidations catalyzed by radical SAM enzymes
(see Scheme 2C).23,24 In these reactions, SAM not only serves
as a radical initiator but also acts as a two-electron oxidant (see
Scheme 2D) that is consumed in a two-to-one ratio versus
substrate in the oxidation of dethiobiotin (5),16,25,26 octanoyl-
LCP (7),17,27 and coproporphyrinogen III (9)28 or in a one-to-
one ratio in the oxidation of 2-deoxy-scyllo-inosamine (10)22
and cysteinyl residues (12).29
The radical SAM enzyme DesII, responsible for the deami-
nation of TDP-4-amino-4,6-dideoxy-D-glucose to give TDP-3-
keto-4,6-dideoxy-D-glucose (15 f 16, Scheme 3) in the
biosynthetic pathway of desosamine, is a particularly noteworthy
example.19,30 Desosamine (see 17) is a 3-dimethylamino-3,4,6-
trideoxysugar derivative important for the antimicrobial activity
of a number of macrolide antibiotics including erythromycin,
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DesII, a Radical SAM Enzyme
A R T I C L E S
Scheme 3
SAM following the deamination of 15 is an attractive mecha-
nism. However, this hypothesis is tempered by the observation
of [5′-2H]-5′-deoxyadenosine ([5′-2H]-14) formation in the
deuterium transfer experiments with TDP-[3-2H]-4-amino-4,6-
dideoxy-D-glucose ([3-2H]-15), and the net oxidative transfor-
mation of 18 catalyzed by DesII.33 As an alternative, the
deamination reaction may in fact be a redox reaction as well,
driven by the reduction of SAM and consequently requires a
continuous input of reducing equivalents.
To address these questions, we determined the stoichiometry
of SAM consumption versus the conversion of the TDP-sugar
substrates in both the deamination and dehydrogenation reac-
tions. The effect of both a nonbiological as well as a biological
reducing source is also studied due to reports that the degree to
which SAM is consumed may vary with the reducing system
employed.44-48 In addition, the ability of DesII to regenerate
the reduced [4Fe-4S]1+ cluster during each catalytic cycle in
the absence of an external source of reducing equivalents is
also investigated. These results are summarized herein, and the
mechanistic implications of DesII catalysis are discussed.
Overall, this study emphasizes the underappreciated redox
capability of SAM in enzyme catalysis (Scheme 2D).1,49 The
general methods reported in this paper should be applicable for
determining the stoichiometry of many other radical SAM
enzymes.
methymycin and pikromycin.31,32 DesII, which contains the
CxxxCxxC consensus sequence, has been verified to be a radical
SAM enzyme on the basis of its dependence on SAM (1) and
a reduced [4Fe-4S] cluster for enzymatic activity.30,33 Con-
sistent with the established mechanisms for this class of
enzymes, the DesII reaction is initiated with a hydrogen atom
abstraction by the 5′-deoxyadenosyl radical (4). Using [3-2H]-
15 as substrate, the transfer of the C-3 hydrogen atom from 15
to the C-5′ position of SAM and/or 5′-deoxyadenosine (14)
during turnover has been demonstrated.33 Subsequent radical
mediated deamination of intermediate 22 (shown in Scheme 5)
to generate 16 may involve a 1,2-migration of the amino group
(from C-4 to C-3), reminiscent of the catalytic mechanisms of
ethanolamine ammonia lyase34-37 and the dioldehydratases.9,38-43
The latter two enzymes rely on adenosylcobalamin instead of
SAM to facilitate the radical induced 1,2-amino or 1,2-hydroxyl
migration during the conversion of ethanolamine or ethylene
glycol to acetaldehyde. DesII, therefore, represents an ideal
system for detailed analysis and direct comparison of the
chemistry underlying radical SAM versus adenosylcobalamin
dependent radical mediated rearrangements in enzymology.
In addition to the deamination of its biological substrate 15,
DesII can also catalyze the two-electron oxidation of the
nonphysiological substrate TDP-D-quinovose to TDP-3-keto-
6-deoxy-D-glucose (18 f 19, Scheme 3).33 This dehydrogena-
tion activity makes the chemistry of DesII catalysis unique,
because the enzyme can act as either a redox neutral deaminase
or an oxidative alcohol dehydrogenase depending on the nature
of the substrate. This raises questions as to the overall redox
chemistry in the DesII-catalyzed reaction. In light of the
precedence set by B12-dependent enzymes as well as the radical
SAM isomerases LAM and SPL (Scheme 2A), regeneration of
Experimental Procedures
Materials. The C-terminal His6-tagged DesII from Strepto-
myces Venzuelae was expressed using Escherichia coli BL21
Star (DE3) cells transformed with a pET24b(+) expression
plasmid, which contains the desII gene, and purified aerobically
by Ni-NTA chromatography and FPLC as previously described.33
Isolated enzyme was judged to be >90% pure based on SDS-
PAGE. Following purification, the enzyme was dialyzed into a
storage buffer containing 50 mM KPi (pH 7.5, KOH) and 15%
glycerol, concentrated to approximately 5 mg/mL, flash frozen,
and stored at -80 °C until use. The enzymes DesI from S.
Venezuelae, FLD and FNR from E. coli, and RfbB from
Salmonella typhi were similarly expressed and purified according
to published procedures.33,50-52 Unless otherwise specified, all
chemical reagents were purchased from commercial sources and
used without further purification. TDP-D-quinovose (18),33,53
TDP-D-glucose (20)54 and 5′-deoxyadenosine (14)55 were pre-
pared according to reported procedures. Commercial sodium
dithionite is known to be contaminated with sodium bisulfite,
sodium thiosulfate and sodium sulfide among other decomposi-
(31) Thorson, J. S.; Hosted, T. J., Jr.; Jiang, J.; Biggins, J. B.; Ahlert, J.
Curr. Org. Chem. 2001, 5, 139–167.
(44) Duschene, K. S.; Veneziano, S. E.; Silver, S. C.; Broderick, J. B. Curr.
Opin. Chem. Biol. 2009, 13, 74–83.
(32) Xue, Y.; Zhao, L.; Liu, H.-w.; Sherman, D. H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12111–12116.
(45) Jarrett, J. T. Curr. Opin. Chem. Biol. 2003, 7, 174–182.
(46) Lotierzo, M.; Raux, E.; Tse Sum Bui, B.; Goasdoue, N.; Libot, F.;
Florentin, D.; Warren, M. J.; Marquet, A. Biochemistry 2006, 45,
12274–12281.
(33) Szu, P.-H.; Ruszczycky, M. W.; Choi, S.-h.; Liu, H.-w. J. Am. Chem.
Soc. 2009, 131, 14030–14042.
(34) Babior, B. M. J. Biol. Chem. 1969, 244, 449–456.
(35) Bandarian, V.; Reed, G. H. Biochemistry 2000, 39, 12069–12075.
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5931.
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Mu¨nck, E.; Fontecave, M. J. Biol. Chem. 2002, 277, 13449–13454.
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M. A. J. Am. Chem. Soc. 1972, 94, 275–277.
(49) Wang, S. C.; Frey, P. A. Trends Biochem. Sci. 2007, 32, 101–110.
(50) Szu, P.-H. Ph.D. Thesis, University of Texas at Austin, 2008.
(51) Zhao, L. Ph. D. Thesis, University of Minnesota, 2000.
(52) Zhao, L.; Borisova, S.; Yeung, S.-M.; Liu, H.-w. J. Am. Chem. Soc.
2001, 123, 7909–7910.
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BioChem 2005, 6, 1423–1430.
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A R T I C L E S
Ruszczycky et al.
tion products.56,57 Therefore, dithionite concentrations are
reported based on absorbance measurements of standards at 315
nm using an extinction coefficient of 6900 M-1 cm-1 at this
wavelength.58 Unless otherwise specified, all reactions were
performed in a Coy anaerobic glovebox under an atmosphere
of >98% N2 and ∼1.5% H2 with <1 ppm O2. Anaerobic water
was prepared by first boiling deionized H2O for 1 h and
immediately transferring the boiling water to the glovebox where
it was stirred open to the atmosphere for at least 48 h. Anaerobic
buffer, 100 mM Tris · HCl (pH 8.0), was prepared by one
freeze-thaw vacuum cycle on an anaerobic Schlenck line under
an argon atmosphere and then transferred to the glovebox where
it was stirred open to the atmosphere for at least 48 h. Anaerobic
water was then added to the buffer to compensate for any loss
of volume.
Scheme 4
enzyme converts 1 µmole of 15 to 16 per minute in the presence
of 0.5 mM 15, 0.5 mM SAM and 0.4 mM Na2S2O4 at pH 8.0 and
25 °C.
HPLC Conditions. Unless otherwise specified, all analytical
HPLC experiments were performed using the same method. This
involved a two-solvent system of A, H2O, and B, 1.5 M ammonium
acetate in H2O. An analytical 4 × 250 mm Dionex CarboPac PA1
anion exchange column was employed with a 4 × 50 mm guard
column. The flow rate was 1 mL/min and the detector was set to
267 nm. The column was eluted with a two-phase linear gradient,
2.5 to 15% B in 20 min then 15 to 20% B in 20 min, followed by
a return to 2.5% B in 5 min.
Synthesis of TDP-4-amino-4,6-dideoxy-D-glucose (15). The
DesII substrate 15 was synthesized with nearly quantitative conver-
sion by coupling the transamination of TDP-4-keto-6-deoxy-D-
glucose (21) by DesI with the oxidation of formate by formate
dehydrogenase (ForDH), as shown in Scheme 4. This coupled
reaction drives the transamination equilibrium to completion and
thus, improves the conversion yield from approximately 20% to
nearly quantitative. A 10 mL solution containing 10 mM TDP-D-
glucose (20), 0.3 mM NAD+, 50 mM KPi (pH 7.5, KOH) and ∼5
mg RfbB was incubated for 1 h at 37 °C. The above reaction
mixture was then made 10 mM in glutamate (monosodium salt),
50 mM in NH4Cl, 50 mM in sodium formate, 1 mM in pyridoxal-
5′-phosphate, and ∼3 mg DesI, 100 U glutamate dehydrogenase
(GluDH) and ∼10 U ForDH were added. The reaction was
monitored by HPLC and was typically complete (>95% conversion)
within 5-10 h in the dark at 37 °C. The protein was removed using
YM10 centrifugal filters, and the product purified by FPLC using
a MonoQ 16/10 column. A flow rate of 3 mL/min was used with
the following gradient: 100% H2O isocratic for 3.3 min, 0 to 350
mM NH4HCO3 in 26.6 min, 350 to 500 mM NH4HCO3 in 6.7 min,
500 mM NH4HCO3 isocratic for 13.3 min. The product typically
elutes at 25 min in approximately 5-10 mL. The eluant was
acidified to pH 6 - 7 by purging with CO2 prior to freezing and
lyophilization. Recovery was greater than 50% and the final ratio
of ammonium salts to product was approximately 5 to 1 based on
the assay with GluDH. The ESI-MS of the isolated product gives
clear signals at 547.8 (positive ion mode) and 546.2 (negative ion
mode) consistent with the M + H+ and (M - H+)- ions,
Reconstitution of the DesII [4Fe-4S] Cluster. One day before
reconstitution, approximately 5 mg of DesII in a volume of roughly
1 mL of storage buffer was thawed and dialyzed overnight against
1 L of 100 mM Tris ·HCl buffer (pH 8.0, 10% glycerol) at 4 °C
under an argon atmosphere. The buffer was first deaerated via 2-3
cycles of argon purge (5 min)/vacuum (20 min) immediately prior
to dialysis. Following buffer exchange, the dialysis system was
taken into the glovebox, where the argon atmosphere was removed
just before cycling in the airlock. Once in the glovebox, the contents
of the dialysis bag were transferred to a 2 mL conical vial chilled
to approximately 10 °C, and the solution was diluted to 2 mL with
anaerobic 100 mM Tris ·HCl buffer (pH 8.0). The solution was
kept cold (10 °C) and stirred for 2 h with the cap off to fully remove
any remaining oxygen. This time period was judged sufficient based
on preliminary measurements of deoxygenation using photoreduced
methylene blue.59 Following deaeration, the solution was incubated
for 15 min in 5 mM DTT by adding 100 µL of 100 mM DTT. The
solution was then incubated for 5 min with 1.5 mM SAM by adding
32 µL 100 mM SAM. A 40 mM solution of Fe(NH4)2(SO4)2 in
anaerobic water was then added over 10 min to a final concentration
of 1 mM. Finally, a 40 mM solution of Na2S in 100 mM Tris ·HCl
(pH 8.0) was added over 20 min to a final concentration of 1 mM.
The resulting reconstitution mixture, which had the appearance of
black coffee, was kept cold (10 °C) and stirred for 3 h with the
vial open to the anaerobic atmosphere, then capped and stirred for
an additional 2 h. The mixture was loaded directly onto a 50 mL
Sephadex-G25 column preequilibrated with 100 mM Tris ·HCl (pH
8.0, 1 mM DTT), and eluted with the same buffer at room
temperature. Colored fractions were collected and assayed with
Bradford reagent to ensure the presence of protein. The enzyme
generally eluted in 4-5 mL, and these were concentrated to 2 mL
using YM10 centrifugal filters in the glovebox at room temperature.
The final enzyme concentration was typically 1-2 mg/mL (18-36
µM) for a recovery of ∼60%. The solutions have a brown to olive-
green color and were stored at approximately 10 °C in the glovebox.
Enzyme reconstituted in this manner has similar properties in terms
of iron-sulfur content as that previously reported33 and a specific
activity of 0.2 ( 0.1 U/mg ((standard deviation), where 1 U of
1
respectively, as the neutral monoisotopic mass of 15 is 547. H
NMR spectra were obtained in unbuffered D2O and are consistent
with previous reports.33,50
Stoichiometry of SAM Consumption versus the Turnover
of TDP-4-amino-4,6-dideoxy-D-glucose (15) and TDP-D-quino-
vose (18). Stoichiometry measurements of SAM consumption
versus 15 or 18 conversion during turnover by DesII were made
using the analytical HPLC methodology described above. Reactions
were performed in a volume of 1 mL containing 100 mM Tris ·HCl
(pH 8.0), 2 mM DTT, 0.25-0.5 mM SAM, and 0.25-0.5 mM of
TDP-sugar substrate. In the case of TDP-D-quinovose (18), initial
SAM and substrate concentrations up to 5 mM were also included.
As a source of reducing equivalents, the reactions contained either
0.2-0.4 mM Na2S2O4 or 0.33 mM NADPH, 20 µM FLD and 70
µM FNR, depending on whether dithionite or the FLD/FNR/
NADPH reducing system was being evaluated. Prior to adding
DesII, a 100-µL aliquot was removed and filtered using YM10
centrifugal filters and frozen until assay by HPLC. This aliquot
was used to determine the initial ratio of TDP-sugar substrate to
SAM in the reaction mixture (see Results). DesII was then added
to a concentration of 4 µM. During the reaction, 100-µL aliquots
were removed at appropriate times and filtered using YM10
centrifugal filters to remove the protein. The filtrate of each sample
was frozen until analysis by HPLC. Chromatograms were used to
determine the stoichiometry as well as to monitor the reaction.
Sluggish reactions were accelerated by adding additional reductant
(56) de Carvalho, L. M.; Schwedt, G. Anal. Chim. Acta 2001, 436, 293–
300.
(57) McKenna, C. E.; Gutheil, W. G.; Song, W. Biochim. Biophys. Acta
1991, 1075, 109–117.
(58) Creutz, C.; Sutin, N. Inorg. Chem. 1974, 13, 2041–2043.
(59) Hamlin, P. A.; Lambert, J. L. Anal. Chem. 1971, 43, 618–620.
9
2362 J. AM. CHEM. SOC. VOL. 132, NO. 7, 2010

DesII, a Radical SAM Enzyme
A R T I C L E S
Figure 1. Representative HPLC chromatograms for stoichiometry experiments with Na2S2O4 as the reductant. (A) Deamination of TDP-4-amino-4,6-
dideoxy-D-glucose (15). (B) Dehydrogenation of TDP-D-quinovose (18). The bottom chromatogram in both (A) and (B) corresponds to the reaction before
adding DesII. Subsequent traces are at increasing incubation times not exceeding 90 and 130 min, respectively. Peaks 14, 16 and 19 correspond to reaction
products 5′-deoxyadenosine, TDP-3-keto-4,6-dideoxy-D-glucose and TDP-3-keto-6-deoxy-D-glucose, respectively. Peaks a, b and c are contaminants in the
commercial SAM. Peak d is a contaminant in the TDP-D-quinovose (18) and corresponds to deoxythymidine monophosphate. Peaks a and 14 overlap, and
the correction for fSAM is described in the Results and further detailed in the Supporting Information.
or DesII, which did not affect the measurement (see Results for
justification). Due to the relative instability of the products and
SAM, reactions were run no longer than 2-3 h. Reactions typically
reached over 60% conversion in the limiting reactant during the
period of the experiment, which increased to >90% on longer
incubations with additional Na2S2O4. Reactions using the FLD/FNR/
NADPH reducing system were typically slower, and lower fractions
of turnover were achieved in the time period of the stoichiometry
experiments. In these cases only experiments that achieved at least
20% conversion of the TDP-sugar substrate were included in the
analysis.
Requirements for a Constant Source of Reducing Equi-
valents. In the case of TDP-4-amino-4,6-dideoxy-D-glucose (15),
a 100 µL solution of 30 µM reconstituted DesII was made 0.4 mM
in Na2S2O4 by adding 10 µL 4 mM freshly dissolved Na2S2O4. The
solution was incubated for 30 min at room temperature and then
15 h at 10 °C. Following the reduction, 10 µL of the reduced DesII
was added to 90 µL of 100 mM Tris ·HCl (pH 8.0) containing 2
mM DTT, 0.5 mM SAM and 0.5 mM of 15. As a control for DesII
inactivation during the 15-h incubation with Na2S2O4, a second
aliquot of 10 µL of the reduced DesII was added to 90 µL of the
same solution that also included 0.4 mM fresh Na2S2O4. Following
the addition of the reduced enzyme, 50-µL aliquots were filtered
to remove protein at 5 and 85 min, and frozen prior to analysis by
analytical HPLC. This experiment was performed twice on two
separate days.
An analogous experiment was performed with TDP-D-quinovose
(18) using the same preparation of DesII following the 15 h
reduction with Na2S2O4. In this experiment a control was also
included to ensure that all of the Na2S2O4 had either been consumed
in the reduction of DesII or decomposed during the overnight
incubation. This control involved filtration of the reduced DesII
enzyme following the 15 h incubation with Na2S2O4 using YM10
centrifugal filters. The previously described reaction system where
15 is replaced with 18 was then treated with 10 µL of 30 µM
nonreduced DesII and 10 µL of the filtrate so as to be in proportions
equal to those used in the experimental sample. Following addition
of the nonreduced enzyme and filtrate, 50-µL aliquots were filtered
to remove protein at 5 and 85 min, and frozen prior to analysis by
analytical HPLC. In tandem with substrate 15, these experiments
with substrate 18 were performed twice on two separate days.
matograms with Na2S2O4 as the reducing agent is shown in
Figure 1A. Approximate retention times are as follows: 5′-
deoxyadenosine (14) at 2.3 min, 15 at 10.0 min, SAM at 17.0
min and TDP-3-keto-4,6-dideoxyglucose (DesII product, 16) at
30.2 min. Additional peaks observed at 2.5 (peak a), 3.0 (peak
b) and 19.5 min (peak c) are due to contaminants in the
commercial SAM. Chromatograms from experiments using
FLD/FNR/NADPH as the reducing system are identical to those
shown in Figure 1A with the addition of a broad, poorly defined
peak at approximately 36 min corresponding to NADP+.
NADPH elutes at a much higher ionic strength than that
employed in the HPLC program and thus is not observed. As
can be seen from the chromatograms in Figure 1A, the peaks
from the contaminants (except for the one at 2.5 min, see below)
neither overlap significantly with those from the reactants and
products nor change during the experimental time period.
Control reactions were run to >99% conversion in both SAM
and 15 demonstrating no significant contaminants underlying
these peaks.
Stoichiometry measurements were performed by monitoring
changes in fraction of reaction for 15, fsub, versus that for SAM,
fSAM. These are each determined per chromatogram by dividing
the area of the product peak by the sum of areas for the product
and corresponding reactant peaks. By doing so, the measure-
ments are insensitive to small variations in the HPLC injection
volumes as well as adjustments to the reaction system, such as
the addition of additional enzyme or reductant, so long as no
additional SAM or 15 is added.
For each experiment an initial chromatogram was obtained
for the reaction system containing all components except DesII,
which was added last to initiate the reactions. This chromato-
gram is used to determine two correction factors for each
experiment. The first, denoted γ, indicates the initial relative
amounts of 15 versus SAM in the reaction,
SAM
267
a0sub
a0SAM
ε
γ )
·
(1)
sub
267
ε
Results
where a0sub and aS0AM are the peak integrations for 15 and SAM,
sub
respectively, in the initial chromatogram, and εS26A7M/ε is the
Stoichiometry of SAM Consumption versus TDP-4-amino-
4,6-dideoxy-D-glucose (15) Conversion. Stoichiometry measure-
ments were made by comparing the relative peak integrations
of the corresponding signals from the analytical ion exchange
HPLC chromatograms. A set of representative HPLC chro-
267
ratio of extinction coefficients for SAM and 15 at 267 nm. The
sub
ratio εS2A67M/ε is found to be 1.525 based on diode-array UV-vis
267
spectra from adenosine and thymidine and using literature values
ado
thy
of ε
) 16982 M-1 cm-1 and ε
) 9332 M-1 cm-1
,
260
269
9
J. AM. CHEM. SOC. VOL. 132, NO. 7, 2010 2363

A R T I C L E S
Ruszczycky et al.
S2SAM - S2sub + ((SS2AM - Ss2ub)2 + 4S2SAM,sub
)
1/2
respectively, to normalize the concentrations. The second
correction factor, denoted κ, is used to remove the contribution
from a small contaminating peak (peak a) that coelutes with
5′-deoxyadenosine (14) and is determined according to
ꢀ )
(5)
(6)
2SSAM,sub
R ) mSAM - ꢀmsub
where S2SAM and mSAM are the sample variance and mean for
the measured values of fSAM, S2sub and msub are the corresponding
values for fsub, and SSAM,sub is the sample covariance of fSAM
κ ) 1 + a0cont/a0SAM
(2)
where a0cont is the area of this contaminating peak in the initial
chromatogram. Assuming the relative intensity of this peak
versus the sum of those from SAM and 5′-deoxyadenosine does
not change over the course of the experiment, then the true
and fsub.
60,61 The slope of the fitted line in any given experiment
is not equal to the stoichiometry, x, because fractions of reaction
are being considered rather than absolute concentrations and
the initial concentrations of SAM and 15 are not precisely equal
in any given experiment. The stoichiometry is instead given by
the ratio
fraction of reaction for SAM, fSAM, can be calculated from the
obs
observed value, f
, as
SAM
obs
SAM
fSAM ) 1 - κ(1 - f
)
(3)
x ) ꢀ/γ
(7)
for which a detailed derivation and explanation is provided in
the Supporting Information.
A detailed explanation and derivation of this correction is
provided in the Supporting Information.
To determine the stoichiometry, x, of SAM versus 15,
Three measurements of the stoichiometry with 15 were
performed for each reducing system. Variances and errors are
determined based on the overall samples of three measurements
each. For the dithionite reducing system, the mean value of x
is 0.96 with a standard deviation of 0.08 and a standard error
of 0.05. For the FLD/FNR/NADPH reducing system, the mean
value of x is 1.05 with a standard deviation of 0.08 and a
standard error of 0.05. These stoichiometries are not significantly
different based on a two-tailed Student’s t-test using a 95%
confidence interval.
Substrate + xSAM DfesII Product + xMet + x5′dAdo
(4)
the fraction of reaction fSAM is plotted against that for fsub, as
shown in Figure 2A and B for representative experiments with
each of the two reducing systems. These plots cannot be fit using
standard least-squares linear regression, because there is error
in both fSAM and fsub. This can be accounted for by assuming
equal variance in both variates and determining the slope, ꢀ,
and ordinate intercept, R, of the fitted line according to
Stoichiometry of SAM Consumption versus TDP-D-quino-
vose (18) Conversion. Stoichiometry measurements of SAM
versus 18 were made in a fashion analogous to that of SAM
Figure 2. Representative fits of the fraction of reaction in terms of SAM, fSAM, versus TDP-4-amino-4,6-dideoxy-D-glucose (15) or TDP-D-quinovose (18),
fsub. Trend lines are fit under the assumption of equal variance in both fSAM and fsub. The stoichiometry is equal to the slope divided by the correction factor,
γ, for the given experiment. (A) Substrate 15 with Na2S2O4 (γ ) 1.23). (B) Substrate 15 with FLD/FNR/NADPH (γ ) 1.05). (C) Substrate 18 with Na2S2O4
(γ ) 1.16). (D) Substrate 18 with FLD/FNR/NADPH (γ ) 2.04).
9
2364 J. AM. CHEM. SOC. VOL. 132, NO. 7, 2010

DesII, a Radical SAM Enzyme
A R T I C L E S
Figure 3. HPLC experiments demonstrating a requirement for a constant source of reducing equivalents in the deamination of TDP-4-amino-4,6-dideoxy-
D-glucose (15) to TDP-3-keto-4,6-dideoxy-D-glucose (16) by DesII. (A) Incubation of reduced DesII with 15 at 5 and 85 min demonstrating less than 2%
turnover at each time point. (B) Incubation of reduced DesII with 15 in the presence of fresh Na2S2O4 at 5 and 85 min demonstrating 23 and 94% turnover
in terms of 15, respectively. Peak 14 corresponds to 5′-deoxyadenosine generated as SAM is consumed. Peaks a and b correspond to contaminants in the
commercial SAM. See text for details and Figure 4 for a comparison with the dehydrogenation reaction.
versus 15. A set of representative HPLC chromatograms using
Na2S2O4 as the reducing system is shown in Figure 1B. These
chromatograms are similar to those observed for the reaction
with 15 except that peaks at 10.0 and 30.2 min are replaced
with peaks at 26.0 and 31.5 min, which correspond to substrate
18 and the TDP-3-keto-6-deoxy-D-glucose (19) product, re-
spectively. A small peak (peak d) eluted just prior to SAM,
which corresponds to deoxythymidine monophosphate (TMP,
present as a contaminant in 18), is well resolved, separable and
less than 5% relative to the initial area of the SAM peak. As in
the case of 15, control reactions were run to >99% conversion
of 18, demonstrating no significant contaminants underlying the
peak corresponding to 18.
While five measurements of the stoichiometry were performed
for the Na2S2O4 experiment, only a single measurement was
performed using FLD/FNR/NADPH as the reducing system. Fits
of fSAM versus fsub for 18 were obtained as described above for
15, and examples using both reducing systems are shown in
Figure 2C and D. Variances and errors are determined for the
Na2S2O4 reducing system based on the overall sample of five
measurements. For the dithionite reducing system the mean
value of x is 1.01 with a standard deviation of 0.11 and a
standard error of 0.05. For the single measurement with the
FLD/FNR/NADPH reducing system, the observed value of x
is 0.97. The stoichiometries between substrates 15 and 18 using
Na2S2O4 as the reducing system are not significantly different
based on a two-tailed Student’s t-test using a 95% confidence
interval.
Additional Controls for Uncoupled Turnover of SAM. Con-
trol reactions were also performed to check for the ability of
DesII to consume SAM in the absence of 15 or 18. Three
reactions were run anaerobically at room temperature (∼25 °C)
in the glovebox in 100 mM Tris ·HCl (pH 8.0), 10% glycerol,
2 mM DTT and 0.2 mM freshly prepared Na2S2O4. The first
reaction included approximately 5 mM 18 and SAM as well as
12 µM DesII to ensure the enzyme was active. The second
reaction included 5 mM SAM and 12 µM DesII but no TDP-
sugar substrate. Aliquots of this reaction were assayed by HPLC
at 0.5, 7, and 48 h (data not shown). On the basis of peak
integrations, 60% of the SAM was decomposed by 48 h,
indicative of a half-life of approximately 35 h. This is compatible
with reported nonenzymatic decomposition of SAM to form
homoserine lactone and 5′-methylthioadenosine by one pathway
as well adenine and S-ribosylmethionine by another, where the
combined half-life at pH 7.5 and the elevated temperature of
37 °C is estimated to be ∼17 h.62 At the retention time of 5′-
deoxyadenosine (14), a small increase in the size of the
contaminating peak was observed; however, this is found to
account for only ∼8% of the decrease in the SAM peak. In
contrast, there was a significant increase in a peak with retention
time similar to adenine at ∼3 min. The increase in this peak
accounts for over 95% of the decrease in the SAM peak,
assuming similar extinction coefficients at 267 nm. In the final
control approximately 5 mM SAM was incubated in the absence
of both DesII and TDP-sugar substrate. In this case it was also
found that the estimated half-life of SAM was approximately
35 h, there was marginal increase in the peak at the retention
time of 5′-deoxyadenosine (14) and the majority of SAM
decomposition was accounted for by an increasing peak with a
retention time similar to that of adenine. Therefore, no significant
differences were observed compared to the reaction in the
presence of DesII.
Requirements for a Constant External Source of Reducing
Equivalents. Reconstituted DesII that had been incubated
overnight with an excess of Na2S2O4 is unable to catalyze
appreciable turnover of either 15 or SAM. This is shown in
Figure 3A where <2% conversion of 15 is observed after 85
min incubation with the reduced DesII in the absence of any
fresh reductant. However, if fresh Na2S2O4 was added in a
concentration equal to that at the beginning of the overnight
incubation, turnover of both SAM and 15 was observed. This
is shown in Figure 3B where 23 and 94% conversion of 15 are
noted at 5 and 85 min, respectively. This indicates that DesII is
still active and the [4Fe-4S] cluster remains intact following
the overnight incubation with the strong reductant.
In contrast, when 18 was incubated with the reconstituted
and reduced DesII in the absence of any additional fresh
Na2S2O4, turnover of both 18 as well as SAM was observed.
As shown in Figure 4A, conversions of 26 and 55% in terms
of 18 were observed at 5 and 85 min, respectively, the reaction
being limited by a substoichiometric amount of SAM. As shown
in Figure 4B, when the deproteinized filtrate from the reduced
(60) Keeping, E. S. Introduction to Statistical Inference; Dover Publications,
Inc.: New York, 1995.
(61) Mandel, J. The Statistical Analysis of Experimental Data; Dover
Publications, Inc.: New York, 1984.
(62) Iwig, D. F.; Booker, S. J. Biochemistry 2004, 43, 13496–13509.
9
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A R T I C L E S
Ruszczycky et al.
Figure 4. HPLC experiments demonstrating that a constant external source of reducing equivalents is not required for the dehydrogenation of TDP-D-
quinovose (18) to TDP-3-keto-6-deoxy-D-glucose (19) by DesII. (A) Incubation of reduced DesII with 18 at 5 and 85 min demonstrating 26 and 55%
reaction in terms of 18, respectively (SAM is substoichiometric versus 18 in this experiment). (B) Incubation of nonreduced DesII with 18 in the presence
of deproteinized reduced DesII filtrate at 5 and 85 min demonstrating less than 1% reaction at each time point. Peak 14 corresponds to 5′-deoxyadenosine
generated as SAM is consumed. Peaks a and b correspond to contaminants in the commercial SAM. See text for details and Figure 3 for a comparison with
the deamination reaction.
DesII was incubated with 18 along with DesII that had not been
treated overnight with Na2S2O4, less than 1% conversion of both
SAM and 18 was observed at 85 min.
seen, indicating that uncoupling remains insignificant and SAM
consumption is not facilitated by the presence of product at the
concentrations and time scale considered. Likewise, only a
marginal increase in 5′-deoxyadenosine (14) is observed in the
control reaction where DesII is incubated with Na2S2O4, DTT
and SAM in the absence of TDP-sugar substrate, and a similar
observation is also noted in the absence of DesII. In these
controls, the majority of SAM consumption is best attributed
to the known nonenzymatic decomposition to adenosine and
S-ribosylmethionine, and/or to homoserine lactone and 5′-me-
thylthioadenosine.62,66 Most importantly, deuterium incorpora-
tion into 5′-deoxyadenosine (14) has been observed in the DesII
reaction with TDP-[3-2H]-4-amino-4,6-dideoxy-D-glucose ([3-
2H]-15),33 presenting a direct link between SAM reduction and
the chemical step of substrate turnover. Together these results
argue against the observed stoichiometry in the deamination
reaction being simply a result of an uncoupling process.
Further support is provided by comparison with the DesII
catalyzed dehydrogenation of 18. As this reaction involves an
overall two-electron oxidation of the TDP-sugar substrate, a
stoichiometry of 1 is expected for the consumption of SAM
versus 18. Indeed, the observed stoichiometry is 1.01 ( 0.05,
and there is no subjective evidence of curvature in the plots of
fSAM versus fsub as seen in Figure 2C and D. This is consistent
with the results from the deamination reaction, arguing against
uncoupling as the cause of SAM depletion. The one-to-one
consumption of SAM per equivalent of 18 dehydrogenated by
DesII is well within the expected behavior of the oxidizing
radical SAM enzymes such as BtrN,22 HemN,28 anSMEs,29 and
the sulfur insertion enzymes (see Scheme 2).16,17,25-27
Discussion
In its biological context, DesII catalyzes the deamination of
15 to 16 in the biosynthesis of TDP-D-desosamine.30,52 As there
is no net redox change in the biologically relevant transformation
of 15 to 16, one would expect SAM to be regenerated with
each catalytic cycle as has been observed with LAM5,10,11 and
SPL.12,13 Therefore, the observation that one equivalent of SAM
is reduced to L-methionine (3) and 5′-deoxyadenosine (14) for
each equivalent of 15 deaminated is unusual both with respect
to other radical SAM mutases and lyases as well as the B12-
dependent lyase EAL and the dioldehydratases.
A recognized aspect of radical SAM enzymology is the
uncoupling of homolytic cleavage of SAM from formation of
the substrate radical.44 This phenomenon is characterized by
the one-electron reduction of SAM by the reduced [4Fe-4S]1+
cluster to methionine (3) and a 5′-deoxyadenosyl radical (4)
that is further reduced to 14 by hydrogen atom transfer or proton
coupled electron transfer from a source other than the nominal
substrate. In the case of biotin synthase, reductive cleavage of
SAM by [4Fe-4S]1+ has been noted in the absence of the
dethiobiotin substrate (5).47 Thus, stoichiometry of SAM
consumption versus biotin (6) production has been reported to
range from 2.1 to 3.1.14,26,46,48 The stoichiometries of turnover
for biotin synthase26 and lipoyl synthase27 are also known to
increase beyond 2.0 with increasing of fraction of reaction.
Similar observations have been made with SPL63,64 and anaero-
bic ribonucleotide reductase,65 where in the latter system the
uncoupled reaction is noted to require the presence of DTT.
In the case of DesII, all fits are subjectively linear with both
substrates as shown in Figure 2. Had there been a significant
uncoupling side reaction, one would expect dependence of the
slope on fsub leading to an upward curvature. Even with reactions
reaching over 75% conversion in 15, no apparent curvature is
While the occurrence of uncoupled reduction of SAM in the
presence of 15 seems unlikely, this does not necessarily rule
out an abortive process leading to the net consumption of SAM.
A scenario can be envisioned where in the deamination of 15
in ViVo, SAM is regenerated with each catalytic cycle analogous
to LAM and SPL. However, rapid, premature reduction of the
[4Fe-4S]2+ cluster may take place in Vitro with the strong
dithionite reductant. In this situation, reduction of [4Fe-4S]2+
by dithionite may occur after initial formation of the 5′-
deoxyadenosyl radical (4) but before hydrogen atom abstraction
from 5′-deoxyadenosine (14) by the product radical. While the
(63) Pieck, J. C.; Hennecke, U.; Pierik, A. J.; Friedel, M. G.; Carell, T.
J. Biol. Chem. 2006, 281, 36317–36326.
(64) Rebeil, R.; Nicholson, W. L. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
9038–9043.
(65) Padovani, D.; Thomas, F.; Trautwein, A. X.; Mulliez, E.; Fontecave,
M. Biochemistry 2001, 40, 6713–6719.
(66) Hoffman, J. L. Biochemistry 1986, 25, 4444–4449.
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DesII, a Radical SAM Enzyme
A R T I C L E S
Scheme 5
5′-deoxyadenosyl radical (4) (and the one-electron reduced SAM
(2)) can still be regenerated at the end of the catalytic cycle,
regeneration of SAM via one-electron reduction of the already
reduced [4Fe-4S]1+ cluster by 2 would not be possible. The
nascent 2 and/or 4 radical may thus be quenched via electron
transfer from dithionite via the iron-sulfur center or hydrogen
atom transfer from an external source such as DTT. The net
result would be an artificial one-to-one stoichiometry.
To test this hypothesis, reconstituted DesII in the [4Fe-4S]2+
state was reduced by overnight incubation with Na2S2O4, during
which time DesII was fully reduced and excess dithionite
decomposed via disproportionation.56 Upon subsequent incuba-
tion with substrate and SAM, significant turnover was observed
only in the dehydrogenation of 18 and not in the deamination
of 15 (Figures 4A and 3A, respectively). These two experiments
likewise control for one another ensuring that the reductant was
exhausted, the cluster was not destroyed, and DesII remained
in the reduced [4Fe-4S]1+ state. Our data also showed that
incubation of 18 with DesII in the oxidized [4Fe-4S]2+ state
mixed with the deproteinized filtrate from the overnight reduc-
tion (in which dithionite had already decomposed) failed to react
(Figure 4B), whereas incubation of 15 with the reduced DesII
and freshly prepared dithionite demonstrated clear turnover
(Figure 3B). Taken together, these results indicate that an
external reducing source is required in the deamination of 15,
implying that consumption of SAM is not an artifact of the
ultimate source of reducing equivalents. Furthermore, the results
with 18 are consistent with either direct or indirect reduction
of the oxidized [4Fe-4S]2+ cluster by the product radical (29/
30 in Scheme 7) in the formation of 19.
To test for an abortive process due to dithionite, the biological
FLD/FNR/NADPH reducing system was considered. This
system is also of interest as it has been suggested that, in the
reaction catalyzed by biotin synthase, changes in the reducing
system employed may lead to slight variations in the observed
stoichiometry.67 However, a one-to-one consumption of SAM
by DesII during deamination of 15 is again observed when FLD/
FNR/NADPH was used as the source of reducing equivalents.
A similar result is also obtained with the dehydrogenation of
18, though only one trial was attempted once it was discovered
that an external source of reductant is not required with this
substrate (see below). These results indicate that in a more
biologically relevant context the DesII enzyme clearly facilitates
deamination of 15 by the net reduction of SAM with a
stoichiometry not significantly different from that with dithionite.
The above findings revealed that, contrary to expectations,
SAM is clearly not regenerated with each deamination reaction.
In this situation, a continuous supply of external reducing
equivalents is required for the net reduction of SAM to complete
the catalytic cycle, because deamination of 15 is redox neutral.
In contrast, the dehydrogenation of 18 should have no such
requirement, since the reducing equivalents would come from
the substrate itself as it is oxidized. Such a mechanism has been
proposed for the radical SAM dehydrogenase BtrN (see Scheme
2C).22 If correct, this hypothesis implies that in the absence of
any external reducing source DesII should only be active in
Vitro for the dehydrogenation reaction so long as DesII is primed
to the [4Fe-4S]1+ state prior to introducing substrate.
On the basis of these observations, DesII may represent a
unique example of a radical SAM enzyme with dual redox
capability.68 The enzyme is able to catalyze both a two-electron
oxidation of 18 and a redox neutral deamination of 15, while
requiring SAM as a two-electron oxidant in both instances.
Mechanistic models for these two modes of DesII catalysis are
proposed in Schemes 5-7. During deamination, formation of
the ternary complex between reduced DesII, SAM and 15
facilitates one-electron reduction of SAM by the [4Fe-4S]1+
cluster triggering homolysis of the C5′-S bond as shown in
Scheme 1. The 5′-deoxyadenosyl radical (4) then abstracts the
C-3 hydrogen atom from 15 completing the two-electron
reduction of SAM (see Scheme 2D) in a manner directly coupled
to the production of the substrate radical 22. In the mechanism
of Scheme 5, L-methionine (3) and 5′-deoxyadenosine (14) are
(67) Farrar, C. E.; Jarrett, J. T. Biochemistry 2009, 48, 2448–2458.
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A R T I C L E S
Ruszczycky et al.
Scheme 6
Scheme 7
the byproduct, and their dissociation may occur at any subse-
quent point in the catalytic cycle.
The resulting ketyl radical (23/24) could then expel ammonia
by an E1cb-type elimination to provide the enol/R-keto radical
25. As an alternative to the E1cb-type elimination from a ketyl
radical, direct, via a 1,2-shift of the amino group, or indirect,
via dissociation/reassociation of ammonia, migration of the
amino group may take place to form 26 as shown in Scheme 6.
These latter possibilities are similar to the current hypotheses
regarding B12-dependent mutases, which are implicated by
computational investigations.70
The fate of the substrate radical is uncertain at this point.
However, as shown in Scheme 5 one possibility is deprotonation
of the C-3 hydroxyl group facilitated by the C-3 radical (22 f
23), consistent with the ability of a carbon-centered radical to
decrease the pKa of an adjacent hydroxyl moiety by 4-5 units.69
(68) The present experiments were conducted with a C-terminal His6-tagged
enzyme. As the native DesII has not been characterized and no detailed
structural information is presently available, the possibility that the
native enzyme may behave differently cannot be excluded.
(69) Hayon, E.; Simic, M. Acc. Chem. Res. 1974, 7, 114–121.
(70) Semialjac, M.; Schwarz, H. J. Am. Chem. Soc. 2002, 124, 8974–8983.
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DesII, a Radical SAM Enzyme
A R T I C L E S
In any case, the radical intermediate (25 or 26) is one-electron
more oxidized than the substrate and product, such that a one-
electron reduction is required to complete the catalytic cycle.
In Schemes 5 and 6, this is proposed to take place via the
[4Fe-4S]1+ cluster, consistent with the ability of the cluster to
oxidize the product radical in the dehydrogenation of 18 (see
Scheme 7). The point at which the [4Fe-4S]2+ cluster is reduced
back to the 1+ state in Schemes 5 and 6 is uncertain and could
conceivably occur at any point following the reductive homoly-
sis of SAM. Protonation of the resulting enolate concomitantly
or in a stepwise manner with regards to electron transfer then
completes the catalytic cycle of the ketyl radical mechanism
(Scheme 5). In the case of the amino-migration type mechanism
(Scheme 6), the net transfer of a hydrogen atom would yield
the carbinolamine 27 that subsequently eliminates ammonia to
afford the ketone product, 16. Reduction of the iron-sulfur
cluster then completes the catalytic cycle.
observation that the immediate precursors to the deamination
reaction, such as 15, 20 and 21, are also precursors to a wide
variety of modified monosaccharides.71 Because the net two-
electron reduction of SAM is expected to be a thermodynami-
cally favorable process, which is consistent with the observation
of virtually quantitative dehydrogenation of 18, coupling deami-
nation of 15 with reduction of SAM would lead the DesII
catalyzed deamination reaction to function as a first-committed,
flux-controlling step in the biosynthesis of TDP-D-desosamine
(17). Investigation of these and other questions pertaining to
the mechanism of DesII are currently a focus of efforts in our
laboratory.
Abbreviations: 5′dAdo, 5′-deoxyadenosine; anSME, anaero-
bic sulfatase maturating enzyme; DTT, dithiothreitol; EAL,
ethanolamine ammonia lyase; FLD, flavodoxin; FNR, flavodoxin
NADP+ oxidoreductase; ForDH, formate dehydrogenase; FPLC,
fast protein liquid chromatography; Glu, glutamate; GluDH,
glutamate dehydrogenase; HemN, oxygen-independent copro-
porphyrinogen III oxidase; HPLC, high performance liquid
chromatography; R-KG, R-ketoglutarate; KPi, potassium phos-
phate; LAM, lysine 2,3-aminomutase; LCP, lipoyl carrier
protein; Met, L-methionine; NAD+, nicotinamide adenine di-
nucleotide (oxidized form); NADH, nicotinamide adenine
dinucleotide (reduced form); NADP+, nicotinamide adenine
dinucleotide phosphate (oxidized form); NADPH, nicotinamide
adenine dinucleotide phosphate (reduced form); PLP, pyridoxal-
5′-phosphate; SAM, S-adenosyl-L-methionine; SPL, spore pho-
toproduct lyase; TDP, deoxythymidine diphosphate; TMP,
deoxythymidine monophosphate.
In the dehydrogenation of 18, generation of the substrate
radical (28) is proposed to take place via an analogous process.
However, rather than eliminating water in place of ammonia,
the anionic substrate radical 29/30 directly or indirectly transfers
an electron back to the oxidized [4Fe-4S]2+ cluster, completing
the net two-electron oxidation of 18 and regenerating the reduced
form of the enzyme. This mechanism likely involves the
deprotonation of the C-3 hydroxyl group of the neutral substrate
radical 28, because the redox potential of a RR′C•sO- radical
is significantly reduced compared to the corresponding
RR′C•-OH radical.69 This process may also be facilitated by
the inability of the enzyme to stabilize a hydroxyl versus an
amino leaving group at C-4 due to the typical 10- to 15-unit
difference in pKa between a secondary hydroxonium and
ammonium cation. This is in contrast with deamination of 15
where elimination of ammonia from 24 to form the more stable
enol/R-keto radical 25 proceeds more rapidly than internal
electron transfer to reduce the cluster. Therefore, the combina-
tion of a poor leaving group and strong reductant would leave
the ketyl radical 29/30 either proceeding with dehydrogenation
or reversing back to substrate and SAM, a phenomenon
consistent with deuterium transfer experiments with both DesII
and BtrN.22,33
Acknowledgment. This work was supported by a grant provided
by the National Institutes of Health (GM035906) and a fellowship
award (F32AI082906) from the National Institute of Allergy and
Infectious Diseases to M.W.R. We thank Steven O. Mansoorabadi
and Wei-chen Chang for helpful comments on the manuscript.
Supporting Information Available: Further detail regarding
the analytical theory behind the stoichiometry measurements.
This material is available free of charge via the Internet at http://
pubs.acs.org.
JA909451A
These results raise the question as to why in a biological
context the deamination of 15 would be coupled to the
consumption SAM. A possible explanation comes from the
(71) Thibodeaux, C. J.; Melancon, C. E.; Liu, H.-w. Nature 2007, 446,
1008–1016.
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J. AM. CHEM. SOC. VOL. 132, NO. 7, 2010 2369