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contain 7.8 Æ 0.2 molFe and 7.5 Æ 0.4 mol S per mol protein,
suggesting that the enzyme contains two [4Fe-4S] clusters.
This result is consistent with analysis with the conserved
domain database (CCD), showing that ArsS consists of an N-
terminal radical SAM domain (pfam04055) and a C-terminal
Cys-rich (pfam12345) domain, the latter domain likely binds
an additional [4Fe-4S] cluster. To test this hypothesis, we
changed Cys45 in the conserved CxxxCxxC motif to Ala, and
the resulting C45A mutant was expressed, purified, and
reconstituted as the wild type enzyme. Subsequent analysis
showed the mutant enzyme contained 5.0 Æ 0.5 molFe and
3.7 Æ 0.3 mol S per mol protein, further supporting ArsS
contains two [4Fe-4S] clusters.
We then performed the assay by incubation of the
reconstituted wild type ArsS with SAM and sodium dithion-
ite. Liquid chromatography with high-resolution mass spec-
trometry (LC-HRMS) analysis of the resulting reaction
mixtures clearly revealed the production of 5’-deoxyadeno-
sine (dAdoH), which exhibited a protonated molecular ion at
m/z = 252.1100 (calc. 252.1097, 1.1 ppm error), and this
product was absent in the control reaction with the super-
natant of boiled enzyme (Figure 2A). dAdoH is the reduced
product of 5’-deoxyadenosyl (dAdo) radical, and hence is the
characteristic product of the radical SAM superfamily. It is
noteworthy that among hundreds of radical SAM enzymes
characterized thus far, only one enzyme (that is, TsrM)
catalyzes a non-radical reaction, and this enzyme does not
produce dAdoH.[9] Significant production of dAdoH in ArsS
reaction is consistent with the proposal that ArsS catalysis
involves a dAdo radical.
Figure 2. In vitro analysis of ArsS. A) LC-MS analysis of ArsS reaction,
showing the extracted ion chromatograms (EICs) of [M+H]+ =252.1
(corresponding to dAdoH) for (i) ArsS reaction in the absence of
DMAsIII, (ii) reaction with DMAsIII and other required components, and
(iii) control reaction using the supernatant of boiled ArsS. B) Time-
dependent production of dAdoH and DDMAA in the ArsS reaction.
Assays were performed by incubation of the reconstituted ArsS
(10 mM) with 2 mM SAM, 5 mM dithionite, with or without 1 mM
DMAIII. The assay was terminated by trichloroacetic acid (5% v/v) at
different time points and analyzed by LC-MS. C) EICs of
[M+H]+ =372.1 (corresponding to DDMAA) for (i) ArsS reaction, (ii)
ArsS reaction spiked with the synthetic DDMAA standard, (iii) control
reactions using the supernatant of boiled ArsS, and (iv) control
reaction without SAM. D) ArsS reaction is redox-neutral and dAdo
radical-dependent. Assay 1 was performed by incubating 2 mM SAM,
and 1 mM DMAIII with 10 mM pre-reduced ArsS for 4 h. Assay 2 was
performed similar to Assay 1, but the pre-reduced enzyme was
exchanged into a fresh buffer without dithionite. Assay 3 is similar to
Assay 2, except the enzyme was first treated with 20 mM SAM for 1 h.
Assay 4 is similar to Assay 3, except additional 1 mM dithionite was
added in the reaction. Assay mixtures were treated with trichloroacetic
acid (5% v/v) and 30% H2O2 (10% v/v) before analyzed by LC-MS. It
is noteworthy that H2O2 converted the reduced product of DDMAA
(for example, DDMAAIII and DDTMAA) back into DDMAA. Different
from the assay in Figure 2D, assays in Figure 2B were not treated with
H2O2.
We next performed the ArsS reaction with DMAsIII,
SAM, and dithionite. LC-HRMS analysis of the reaction
mixture showed that, although dAdoH is produced in the
reaction, its yield decreased significantly compared to the
reaction without DMAsIII (Figure 2A, and B). This observa-
tion is in contrast to most radical SAM-dependent reactions in
which dAdoH production is generally increased in the
presence of substrate.[8c] Instead, it is reminiscent of the
recent studies in achieving the dAdo radical-based addition
reactions by using unnatural substrates with an olefin
moiety,[10] suggesting that the dAdo radical may possibly
add to DMAIII in the ArsS-catalyzed reaction. Indeed,
a compound with a protonated molecular ion at m/z =
372.0652 was observed (Figure 2C, trace i) in the ArsS
reaction mixture, and this compound is absent in the negative
control reactions without SAM or with the supernatant of
boiled enzyme (Figure 2C). The suggested molecular formula
C12H18AsN5O4 ([M+H]+ calc. 372.0653, 0.3 ppm error) is
consistent with DDMAA, and this was further supported by
HR-MS/MS analysis, showing a series of fragment ions
characteristic of adenosine-containing compounds (Support-
ing Information, Figure S1).
is a DDMAA synthase, which catalyzes As alkylation via
radical SAM chemistry.
Characterization of ArsS as a DDMAA synthase suggests
that the failure in finding DDMAA in the E. coli expression
system[6] is likely because DDMAA was modified by
unknown enzymes in E. coli cells. To test this proposal, we
incubated DDMAA with E. coli cell lysate, and the resulting
mixture was analyzed by LC-HRMS. This analysis showed
that indeed, DDMAA decomposed substantially in E. coli
lysate, whereas it appeared quite stable in Tris buffer
(Supporting Information, Table S1). However, in our analysis
we did not observe the production of dimethylarsinoyl-
hydroxycarboxylic acids (such as compound 1, 2, and 3)
reported previously,[6] reflecting the difference between in
vivo and in vitro systems. Intriguingly, we found that with E.
coli cell lysate, DDMAA decomposed more completely in an
anaerobic condition (Supporting Information, S1), and it was
(at least partially) converted into an aldehyde compound
(Supporting Information, Figure S2). Future studies are
awaited to reveal the enzymes involved in DDMAA decom-
position in E. coli.
To further validate the identity of this compound,
DDMAA was chemically synthesized and was used as
a reference in LC-HRMS analysis. The results showed that
the synthetic standard was co-eluted with the compound
produced in the reaction (Figure 2C), and both compounds
have exactly the same HR-MS/MS spectra (Supporting
Information, Figure S1). These results strongly support ArsS
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