Article
pubs.acs.org/est
Arsenic Demethylation by a C·As Lyase in Cyanobacterium Nostoc sp.
PCC 7120
Yu Yan,†,‡,∥ Jun Ye,†,∥ Xi-Mei Xue,† and Yong-Guan Zhu*,†,§
†
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021,
People’s Republic of China
‡
University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
§
State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
Beijing 100085, People’s Republic of China
S Supporting Information
*
ABSTRACT: Arsenic, a ubiquitous toxic substance, exists mainly as
inorganic forms in the environment. It is perceived that organoarsenicals can be demethylated and degraded into inorganic arsenic
by microorganisms. Few studies have focused on the mechanism of
arsenic demethylation in bacteria. Here, we investigated arsenic
demethylation in a typical freshwater cyanobacterium Nostoc sp.
PCC 7120. This bacterium was able to demethylate monomethylarsenite [MAs(III)] rapidly to arsenite [As(III)] and also had the
ability to demethylate monomethylarsenate [MAs(V)] to As(III).
The NsarsI encoding a C·As lyase responsible for MAs(III)
demethylation was cloned from Nostoc sp. PCC 7120 and
heterologously expressed in an As-hypersensitive strain Escherichia
coli AW3110 (ΔarsRBC). Expression of NsarsI was shown to confer MAs(III) resistance through arsenic demethylation. The
purified NsArsI was further identified and functionally characterized in vitro. NsArsI existed mainly as the trimeric state, and the
kinetic data were well-fit to the Hill equation with K0.5 = 7.55 ± 0.33 μM for MAs(III), Vmax = 0.79 ± 0.02 μM min−1, and h =
2.7. Both of the NsArsI truncated derivatives lacking the C-terminal 10 residues (ArsI10) or 23 residues (ArsI23) had a reduced
ability of MAs(III) demethylation. These results provide new insights for understanding the important role of cyanobacteria in
arsenic biogeochemical cycling in the environment.
■
INTRODUCTION
Arsenic (As) is a ubiquitous toxic and carcinogenic substance in
the environment.1 Arsenic exposure through food and water are
causing human health problems, including lung and skin
cancers, in many parts of the world.2,3 Arsenic was ranked the
first on the National Priorities List (NPL) of Hazardous
Substances (http://www.atsdr.cdc.gov/SPL/index.html) by the
Agency for Toxic Substances and Disease Registry (ATSDR).
The biotransformations of inorganic arsenic into organoarsenicals are important parts of global arsenic biogeochemical
cycle. Inorganic As, including arsenate [As(V)] and arsenite
[As(III)], are dominant chemical species in the environment.4,5
However, microorganisms can convert inorganic As into
organic species.6 The microbial methylation detoxifies As(III)
by sequentially producing less toxic MAs(V), dimethylarsenate
[DMAs(V)], trimethylarsine oxide [TMAsO(V)], and nontoxic
volatile trimethylarsine [TMAs(III)].7,8 However, MAs(III)
and dimethylarsenite [DMAs(III)] produced in the methylation process are more toxic than As(III) for both
mammalian9,10 and microbial cells.11 As(III) S-adenosylmethionine methyltransferases (ArsMs) from bacteria, archaea,
and eukaryotes (except higher plants) catalyzing the biomethylation have been characterized.12−16 The widespread and
© 2015 American Chemical Society
early-evolved ArsMs continually methylate the environmental
arsenic. In addition, substantial amounts of organoarsenicals,
such as monosodium methylarsonic acid (MSMA), were largely
introduced into the environment by their use as herbicides and
pesticides during the past half century.17 Nonetheless, the
majority of environmental arsenic exists as inorganic forms.
This is because the demethylation of organoarsenicals may be
extensive in both terrestrial and aquatic environments, and
microbes have been found to be involved in As demethylation.18,19 Based on As species analysis, studies have been done
on the transformation of methylarsenicals in the soils of
organoarsenic-contaminated sites, such as cotton fields20 and
golf course greens,21 where high levels of MSMA were used as
herbicides. Moreover, the biodegradation of organoarsenicals
has also been evaluated in anaerobic sludge22 and lakes.23,24
Furthermore, methylarsenic-decomposing microorganisms have
been classified and isolated from soil and water,25 and it has
been shown that only a few types of microbes, such as
Received:
Revised:
Accepted:
Published:
14350
July 11, 2015
September 23, 2015
November 6, 2015
November 6, 2015
DOI: 10.1021/acs.est.5b03357
Environ. Sci. Technol. 2015, 49, 14350−14358
Article
Environmental Science & Technology
Montrachet wine yeast,26 Candida humicola,27 and Mycobacterium neoaurum28 are capable of As demethylation. A novel twostep pathway of MSMA degradation by reducing MAs(V) to
MAs(III) and demethylating MAs(III) to As(III) in sequence
was demonstrated recently.29 The two steps are performed by
different bacterial species isolated from Florida golf course soil.
The gene, namely arsI (As-inducible gene),30 responsible for
the second-step MAs(III) demethylation was identified from
the environmental MAs(III)-demethylating isolate Bacillus sp.
MD1, and the ArsI protein confers MAs(III) resistance and has
Fe2+-dependent C·As lyase activity.11 Its ortholog from
Thermomonospora curvata has been crystallized to determine
the three-dimensional structure.31
Ancient cyanobacteria as photosynthetic prokaryotes are
globally widespread, found from seawater and freshwater
systems to soils and even deserts.32 Cyanobacteria often grow
in heavy metal(loid)-contaminated habitats due to their ability
to resist metal toxicity.33 Previous studies have indicated that
cyanobacteria have multiple As biotransformation pathways
and, in particular, make great contributions to As methylation.14
However, it is still unclear whether cyanobacteria are associated
with the reverse reaction, As demethylation, not to mention the
molecular mechanism. Nostoc sp. PCC 7120 (Nostoc) is a
typical filamentous nitrogen-fixing cyanobacterium. Nostoc has
been extensively studied as a model organism for the genetic
analysis of photosynthetic and nitrogen fixation processes. The
sequencing of the entire genome of Nostoc has been completed
in 2002.34 For As metabolism, Nostoc can methylate As(III)
into methylated arsenicals35 and produce arsenosugars.36
Because of its available genomic and As metabolic information,
we focused on the molecular mechanism underlining As
demethylation by Nostoc in this study. Herein, we investigated
methylarsenical degradation pathway of Nostoc and focused on
the molecular mechanism of As demethylation. By rapidly
demethylating As, cyanobacteria including Nostoc play a
significant role in As biogeochemical cycling. Our results also
shed light on the molecular mechanism of As metabolism in
cyanobacteria.
by centrifugation (6500g, 5 min) at each different time
intervals. The supernatants were filtrated through 0.22 μm
disposable filters and kept at −80 °C until analysis. The cells
were washed with precooled MES buffer [1 mM K2HPO4, 5
mM MES, and 0.5 mM Ca(NO3)2 at pH 6.2]39 to remove
apoplastic As and then lyophilized. The cellular As was
extracted by deionized water (18.2 ΩM cm−1) as described
below. After the appropriate 0.01 g of freeze-dried cells had
been transferred to lysing tubes with 0.5 g glass beads, 300 μL
of deionized water was added into the tubes. Sequentially the
cell lysis was carried out with Fastprep-24 (MP Biomedicals)
for 60 s at a speed of 6.5 m s−1. The cell lysis liquid was
centrifuged (13400g, 1 min) at 4 °C, and then the supernatants
were carefully pipetted into a new tube. The above steps were
repeated three times, and the supernatants were combined and
filtrated through 0.22 μm filters to determine the As species.
Cloning of arsI from Nostoc. To identify the putative arsI
in Nostoc, we used the protein sequence of confirmed ArsI in
Bacillus sp. MD1 (accession no. KF899847.1) as a query to
perform a similar search (BALSTP) against the proteins of
Nostoc. The result showed that alr1104 (accession no.
BAB73061) annotated as glyoxalase was an ortholog of ArsI.
Thus, we amplified alr1104 (named as NsarsI) from the
genomic DNA, which was isolated from Nostoc by using a plant
genomic DNA extraction kit (Bioteke) according to the
manufacturer’s instructions. The NsarsI consisting of the start
codon and excluding the stop codon was amplified by PCR
with primers 5′-CATATGTCCGTTATGAAAACACACG-3′
(NcoI restriction site underlined) and 5′-CTCGAGAGCACAACATGACTTC-3′ (XhoI restriction site underlined). The
PCR products were purified and cloned into plasmid pMD19-T
simple vector (Takara) to result in the plasmid pMD19TNsarsI. After digestion with NdeI and XhoI, NsarsI was cloned
into the NdeI−XhoI-digested expression vector pET22b
(Novagen) to generate plasmid pET22b−NsarsI. The
pET22b−NsarsI was subsequently transformed into the E. coli
Rosetta (DE3) or AW3110 (ΔarsRBC).
Overexpression and Purification of NsArsI in E. coli.
Rosetta cells bearing pET22b−NsarsI were incubated at 37 °C
in LB medium containing ampicillin (100 μg mL−1) and
chloramphenicol (30 μg mL−1). Overnight cultures of E. coli
were diluted 100-fold into 2 L of fresh LB medium with
ampicillin and chloramphenicol. Exponential phase cells were
induced by 1 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) to express His-tagged NsArsI. After being continually
cultured at 16 °C overnight, the cells were harvested by
centrifugation (4 °C, 6000g, 15 min) and resuspended with
HEPES buffer [20 mM HEPES, 500 mM NaCl, 3 mM tris(2carboxyethyl)phosphine (TCEP), pH 7.2]. The cells were
disrupted by a single pass through a French press at 10 MPa
and centrifuged at 4 °C and 13000g for 1 h. The supernatant
was gravity-loaded onto a Ni-NTA agarose column (Qiagen)
pre-equilibrated with HEPES buffer and washed with 20 mM
imidazole in HEPES buffer. The protein was then eluted with
15 mL of HEPES buffer containing 500 mM imidazole, and
further purified by size-exclusion chromatography (HiLoad 16/
600 Superdex 75 pg, GE Healthcare Life Sciences) with HEPES
buffer. The elution volume of NsArsI was compared with the
elution profile of molecular-mass standards (BioRad) to
investigate its oligomer status. The purified NsArsI was
identified by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and concentrated by ultrafiltration (10 kDa cutoff Amicon Ultrafilter, Millipore).
■
MATERIALS AND METHODS
Organisms and Cultivations. Nostoc was kindly provided
by Professor Wen-Li Chen, Huazhong Agricultural University,
and cultivated in BG11 medium without nitrate.37 Experiments
were carried out in a light-equipped shaker incubator under a
12 h dark−light cycle with a light intensity of approximately 50
μmol photons (m−2 s−1) at day and night temperatures of 27
and 25 °C, respectively.
Unless otherwise noted, E. coli strains were grown at 37 °C
aerobically in Luria−Bertani (LB) medium with shaking at 180
rpm. For plasmid construction and replication, E. coli strain
DH5α (Promega) was used. The As(III) hypersensitive E. coli
strain AW3110 (DE3)38 [ΔarsRBC; ArsR-repressor, ArsBAs(III) efflux pump, and ArsC-As(V) reductase] was used for
resistance assay and functional verification of NsarsI. E. coli
strain Rosetta (DE3) (Novagen) was used for protein
expression.
Chemical Reagents. The MAs(III) was prepared by
reduction of MAs(V) as previously described.11 All other
reagents were obtained from commercial sources and of
analytical grade or better.
Arsenic Demethylation by Nostoc. When the cultures
were in the exponential phase, Nostoc was treated with 1 μM
MAs(V) or MAs(III) in triplicate. The samples were harvested
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MAs(III) Resistance Assays in E. coli. MAs(III) resistance
assays were performed as previously described.11 Single colony
of AW3110 (ΔarsRBC) bearing either pET22b or pET22bNsarsI was incubated into 5 mL of LB medium containing
antibiotics with shaking (200 rpm) at 37 °C overnight. The
cells were diluted 100-fold into 30 mL of fresh LB medium with
antibiotics and induced by adding 1 mM IPTG when the
optical density at 600 nm (OD600 nm) reached 0.6−0.8. After
incubation at 16 °C for an additional 18 h, the induced cells
were washed with an equal volume of ST medium (10-fold
concentrated ST 10−1 medium23) and diluted 50-fold into 30
mL of fresh ST medium supplemented with antibiotics and
0.2% D-glucose. The cells were cultured with the indicated
concentrations of MAs(III) at 30 °C with shaking at 200 rpm.
The OD600 nm was monitored by using an ultraviolet−visible
spectrophotometer (UV-6300 double beam spectrophotometer, Mapada).
MAs(III) Demethylation Assays in E. coli. AW3110
(ΔarsRBC) bearing either pET22b or pET22b−NsarsI was
used to measure MAs(III) demethylation. The IPTG-induced
cells were incubated for an additional 6 h and then transferred
to ST 10−1 medium containing the appropriate antibiotics and
0.5 μM MAs(III). After incubation at 25 °C for another 1 h, the
samples were centrifuged at 13400g for 2 min; the supernatants
were filtrated through 0.22 μm filters, the pellets were washed
and lysed with deionized water using the method for
cyanobacteria as described above. The MAs(III)-demethylating
activity was analyzed by determining As species in both the
medium and cells.
MAs(III) Demethylation Assays Using Purified NsArsI.
In vitro reaction of MAs(III) demethylation with 10 μM
purified NsArsI was performed in triplicate in a 100 mM MOPS
buffer (pH 7.0) at 30 °C for 1 h. The reaction mixture also
contained 3 mM TCEP, 1 mM cysteine, 0.1 mM Fe2+, and 10
μM MAs(III). Each assay mixture was immediately terminated
by adding 40 mM EDTA at the indicated time point and
divided into two parts. One portion was directly diluted 10-fold
and filtered through a 0.22 μm filter to monitor the As species
during the reaction process, and the other portion was used to
determine the nature of NsArsI-bound arsenicals as described
below. The samples were passed through BioGel P6 columns
(BioRad) pre-equilibrated with MOPS buffer, followed by
dilution with 6 M guanidine HCl at once to denature protein
and release the bound As, the species of which were
determined. For kinetic assays, the reaction was performed
with the indicated concentrations of MAs(III). The amounts of
As(III) generated at 1 min by 10 μM NsArsI were used as
approximate initial rates. Nonlinear regression analysis was
performed with SigmaPlot 12.5.
MAs(III) Demethylation of NsArsI Truncated Derivatives in Vivo and in Vitro. The results from multiple
sequence alignment of ArsI homologues showed that there is
little conserved sequence in C-termini. In addition, arsI124,
which is an arsI derivative from Bacillus sp. MD1 encoding only
the N-terminal 124 residues still has the C·As lyase activity.11
Herein, two truncated derivatives of NsArsI including ArsI10
and Ars23 were constructed to verified whether the C-terminal
residues of NsArsI influence the MAs(III) demethylation
activity. The forward primer used for truncated derivatives
was the one for full-length NsArsI. The reverse primers were 5′CTCGAGTTCCCCAGAAGAACTTAC-3′ (XhoI restriction
site underlined) and 5′-CTCGAGCGCTGTATCTGCTAC-3′
(XhoI restriction site underlined) for ArsI10 and ArsI23,
respectively. The other conditions for gene cloning, protein
purification, and functional verification in vivo and in vitro of
these derivatives were the same to the full-length NsArsI unless
otherwise indicated.
Arsenic Speciation Analysis. Arsenic speciation was
assayed by high-performance liquid chromatography−inductively coupled plasma mass spectrometry (HPLC−ICP-MS,
7500a, Agilent Technologies) using the previous instrument
parameters.12 A Jupiter 5 μ C18 300A reverse-phase column
(250 mm × 4.6 mm, Phenomenex) was used for arsenic
compounds separation. The mobile phase (pH 5.95) consisted
of 3 mM malonic acid, 5 mM tetrabutylammonium hydroxide,
and 5% methanol at a flow rate of 1.0 mL min−1. The ICP-MS
was tuned for the monitoring of m/z 75 (arsenic). At the same
time, m/z 77 and m/z 82 (selenium) were used for monitoring
the interference by ArCl, and m/z 72 (germanium) and m/z
103 (rhodium) were used as internal standards to compensate
for any instrument drift. Arsenic species in the samples were
identified by retention times, compared with those of the
standards composed of As(III), MAs(III), DMA(V), MAs(V),
and As(V). Arsenic amounts were quantified by external
calibration curves with peak areas.
■
RESULTS
Nostoc Demethylation of Both MAs(III) and MAs(V) to
As(III). Biodemethylation of MAs(III) by Nostoc was measured
during a 24 h period. During the first 12 h, the concentration of
As(III) increased rapidly in the medium, concomitant with the
decrease of MAs(III) (Figure 1A). As(III) concentration in the
medium did not increase beyond 12 h. During the whole
incubation period, MAs(V) was shown to be the major As
species in the cells (Figure S1) and may be the product of
MAs(III) oxidation by enzymatic or nonenzymatic reactions. It
is also possible that the detected MAs(V) was formed by
MAs(III) oxidation during sample preparation. However,
As(III), accounting for only 6 to 25% of the total As in cells,
was relatively less, and its peak appeared in the third hour.
As(V) was also detected in the cells and showed a similar trend
to As(III). There was no inorganic arsenic detected in the
control without Nostoc inoculation. MAs(V) demethylation by
Nostoc was measured during 3 weeks of treatment. The rate of
demethylation for MAs(V) was much slower than that for
MAs(III) (Figure 1B). The increase in the quantity of As(III)
coincided with the decrease of the MAs(V) in the medium
during the initial 15 days, and there was no further increase in
As(III) from 15 to 21 days. Quite a small amount of MAs(III)
was also detected for 1−9 days, accounting for 0.4−1.1% of the
total arsenic in the medium.
Cloning of NsarsI. A putative 450 bp length NsarsI was
yielded by PCR using the genomic DNA of Nostoc. NsarsI
encodes NsArsI of 150 residues, with a predicted molecular
mass of 17.35 kDa. The analysis of sequence alignment showed
that NsArsI had a high degree of sequence homology (45%
identity and 83% similarity) to ArsI from Bacillus sp. MD1.
NsArsI contained the histidine(7)−histidine(65)−glutamic
acid(117) motif as a putative divalent metal (Fe2+) binding
site and a cysteine pair at residues 98−99 as a putative
MAs(III) binding site (Figure S2). Besides, the first residue
(His7) of the putative Fe2+ binding site was not conserved and
was replaced by glutamine residue in the putative ArsI ortholog
from Phycicoccus jejuensis.
E. coli AW3110 Bearing NsarsI Conferment of MAs(III)
Resistance. When NsarsI was expressed in E. coli AW3110,
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Figure 1. MAs(III) and MAs(V) demethylation by Nostoc. (A) As
species in BG11 medium with Nostoc exposed to MAs(III) for the
indicated times (0, 1, 3, 6, 9, 12, and 24 h). (B) As species in BG11
medium with Nostoc exposed to MAs(V) for the indicated times (0, 1,
3, 6, 9, 12, 15, 18, and 21 days). The error bars indicate the standard
error of triplicate assays.
which had neither chromosomal arsRBC nor orthologous arsI,
it conferred resistance to MAs(III) (Figure 2A). When grown
on MAs(III)-free ST medium, AW3110 cells bearing either
pET22b or pET22b−NsarsI showed almost no difference in
growth rate. At 1 and 3 μM of MAs(III), AW3110 expressing
NsarsI grew significantly faster than that bearing pET22b at the
7 to 24 h time points (t test, P < 0.05). AW3110 bearing
pET22b−NsarsI still grew slightly faster than that bearing
empty plasmid at 9 μM of MAs(III), although both of AW3110
were strongly inhibited at higher concentration MAs(III).
MAs(III) Demethylation to As(III) by E. coli AW3110
Expressing NsarsI. To elucidate whether NsarsI has MAs(III)
demethylation activity in vivo, we incubated AW3110
expressing NsarsI in ST medium containing 1 μM MAs(III),
and the products of demethylation reactions were quantified in
the culture medium (Figure 2B). After AW3110 bearing NsarsI
had been cultured for 1 h, MAs(III) became undetected, and
As(III) was the dominant arsenic species in the medium.
Meanwhile, only inorganic arsenic, including As(III) and
As(V), was detected in the cells lysates of AW3110 expressing
NsarsI (Figure 2C). There were trace amounts of As(III)
detected in both the medium and the cells of AW3110 bearing
pET22b. It was probably coming from the slightly contami-
Figure 2. MAs(III) resistance and demethylation by AW3110
(ΔarsRBC) expressing pET22b-NsarsI. (A) The growth curves of
AW3110 bearing pET22b-NsarsI or pET22b. Absorbance was
monitored at 600 nm. (B) As species in ST medium cultured with
AW3110 exposed to 0.5 μM MAs(III) for 1 h at 25 °C. (C) HPLC−
ICP-MS analysis of the As species in the cell lysate of AW3110 as
described in the Materials and Methods section. AW3110 bearing
pET22b was the control.
nated MAs(III) used in the experiments because the initial
substrate also contained a trace amount of As(III).
MAs(III) Demethylation to As(III) by Purified NsArsI in
Vitro. To make the mechanism of the enzyme more
comprehensible, we analyzed changes of arsenic species in
vitro assayed with purified NsArsI over 1 h. Purified His-tagged
NsArsI as soluble protein had a molecular mass of
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approximately 17 kDa, which was basically coincided with the
predicted molecular mass (Figure S3). Once purified NsArsI
was added into the reaction, the quantity of MAs(III) declined
sharply, although there was only a small increase in As(III).
With the reaction time prolonging in 1 h, the concentration of
As(III) continually increased, and total soluble arsenic gradually
recovered to the level without adding NsArsI (Figure 3A).
Figure 4. Possibility of the oligomeric state of NsArsI and the kinetics
of the MAs(III) demethylation reaction catalyzed by NsArsI. (A) The
standard curve for protein molecular mass using analytical sizeexclusion chromatography on a HiLoad 16/600 Superdex 75 column.
The values for Ve of NsArsI shown as red dots are 60.9 and 69.4 mL.
(B) The kinetic curve of NsArsI for MAs(III) demethylation with the
indicated MAs(III) concentrations (0.5, 2, 3, 4, 5, 10, 20, and 40 μM).
value of 2.7 rather than Michaelis−Menten eq (Figure 4B). The
rest kinetic parameters of K0.5 and Vmax for MAs(III) were 7.55
± 0.33 μM and 0.79 ± 0.02 μM min−1, respectively.
Truncated NsArsI Derivatives Were Active. There were
nonconserved residues located in the C-termini of NsArsI, and
the previous studies indicated that removal of these residues in
other As resistance enzymes such as ArsM improve protein
purification and crystallization instead of losing function.41
Therefore, we constructed two truncated NsArsI derivatives
(ArsI10 and ArsI23) to verify whether the C-terminal residues
have an impact on function. When AW3110 expressing ArsI10
or ArsI23 was incubated in the medium with 3 μM MAs(III),
MAs(III) was completely converted to As(III), and the rate was
almost as the same as AW3110 expressing the full-length
NsArsI (ArsIall) (Figure 5A). However, in the in vitro reaction
with the addition of 30 μM MAs(III), the purified ArsI10 or
Ars23 only demethylated 2.1% or 15.6% MAs(III) while ArsIall
was able to demethylate up to 29.6% MAs(III) (Figure 5B).
This suggested that the nonconserved residues, despite not
being critical for MAs(III) demethylation, still had negative
effects on the efficiency of MAs(III) demethylation.
Figure 3. MAs(III) demethylating functional verification of NsArsI.
(A) Changes in As species in MOPS buffer (pH 7.0) for assays of
MAs(III) demethylation by NsArsI at 37 °C over 1 h. (B) HPLC−
ICP-MS analysis of the enzyme-bound arsenicals of NsArsI in vitro
assays under the same conditions in (A) for the indicated times.
Hence, we predicted that MAs(III) was quickly and strongly
bound to the enzyme,40 so arsenic species bound to NsArsI was
analyzed. The results showed that MAs(V) was present in the
entire reaction and might be the product of MAs(III) oxidation
during sample preparation, while As(III) was mainly observed
in the last 40 min (Figure 3B).
NsArsI Mainly Existed as a Trimer. Size-exclusion
chromatography of NsArsI resulted in a main peak at 69.4
mL and a secondary peak at 60.9 mL (Figure S4). The elution
profile of NsArsI corresponded to a mass of 50 kDa and 106
kDa by comparison with molecular-mass standards (Figure
4A). Although the predicted NsArsI monomer mass was about
17 kDa. Thus, NsArsI probably mainly existed as a trimer with a
little mixture of hexamer in solution. In agreement with the
chromatography, the kinetic of NsArsI for MAs(III)
demethylation was in accord with the Hill equation with an h
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important sources of the intracellular MAs(V). Recently, ArsH
was found to be a trivalent organoarsenical oxidase,43 but the
ArsH ortholog was not found in Nostoc, and MAs(III) might be
nonspecifically oxidized by other oxidases. Therefore, ArsI is
considered to be the dominant enzyme involved in MAs(III)
detoxification in Nostoc. Indeed, our results clearly showed that
MAs(III) was almost entirely demethylated to As(III) and then
effluxed to the medium.
MAs(III) demethylation is a pathway to detoxify more
acutely toxic MAs(III) to As(III). MAs(III) in the environment
is mainly generated by ArsM.44 The widespread distribution of
arsMs implies that arsenic methylation occurred on early Earth
even before the atmosphere became oxidizing.1,11 MAs(III) as
the initial product of As methylation was stable in the absence
of oxygen, similar to how the organisms on early Earth must
have evolved MAs(III) resistance mechanisms to protect
themselves. Cyanobacteria with oxygenic photosynthesis were
the earliest important producers of atmospheric oxygen.45,46
When cyanobacteria were under MAs(III) selective pressure,
the evolution of ArsI using self-generated molecular oxygen to
break the C−As bond of MAs(III) seems quite economical and
effective. Although MAs(III) can be rapidly oxidized in the
present oxidizing atmosphere, MAs(III) still exists in the
natural environment. For example, a high concentration of
MAs(III) was found in groundwater contaminated by herbicide
production,47 and MAs(III) could also be detected in
freshwaters.48 In addition, the widely used MASA was
constantly reduced by various organisms to MAs(III) as its
active form.49 Thus, MAs(III) demethylation catalyzed by ArsI
make cyanobacteria stay away from the poison of MAs(III)
throughout the ages.
Because methylation and demethylation are two opposite
arsenic metabolic pathways, it should be noted that two of the
pathways coexist in Nostoc because they have both arsM35 and
arsI genes. After exposure to higher As(III) concentrations
(e.g., 100 μM), Nostoc methylated As(III) to DMAs(V) and
volatile TMAs(III) with ArsM 35 or further produced
arsenosugars with unknown enzymes.36 MAs(III), a predicted
intermediate in the pathway of methylation, was not detected
during As methylation in Nostoc.35 It is likely that the produced
MAs(III) could be quickly demethylated to As(III) by ArsI or
oxidized to MMA(V) by enzymatic or nonenzymatic reactions.
When Nostoc was treated with lower concentrations of As(III)
(e.g., 10 μM), As(III) cannot be methylated but was detoxified
by direct efflux out of cells or oxidation to As(V) as described
by Yin et al.35 The present study suggested that As(III)
produced by demethylation of MAs(III) at 1 μM is not enough
to remethylate to organoarsenicals. In short, cyanobacteria have
evolved versatile detoxification mechanisms to deal with various
arsenic species. Because MAs(III) is more toxic than As(III),
MAs(III) demethylation with higher reaction rate and lower
concentration of substrate than As(III) methylation seems to
be critical for the survival of cyanobacteria in arseniccontaminated environments. Furthermore, MAs(III) is considered as a primitive antibiotic, and cyanobacteria are “smart”
enough to rid themselves of MAs(III) by demethylation.
ArsI is considered a nonheme Fe(II)-dependent extradiol
dioxygenase with C·As lyase activity.11 Our results indicated
that NsArsI exists as a trimer in the solution. It is not surprising
that the extradiol dioxygenases always exist in a range of
oligomeric states;50 for example, gallate dioxygenase from
Pseudomonas putida KT2440 was also a trimer composed of
three identical subunits.51 In accordance with the trimeric state
Figure 5. MAs(III) demethylation of MAs(III) by full-length NsArsI
and truncated derivatives. (A) In vivo MAs(III) demethylation assays
were conducted in the ST medium exposed to 3 μM MAs(III) and
inoculated AW3110 expressing arsIall, arsI10, or arsI23. AW3110
bearing pET22b was used as the control. (B) In vitro assays of
MAs(III) demethylation using purified proteins (ArsIall, ArsI10, and
ArsI23) were tested in MOPS buffer (pH 7.0) containing 30 μM
MAs(III). The reaction solution without adding NsArsI was the
control.
■
DISCUSSION
To our knowledge, NsArsI is the first identified and
characterized MAs(III) demethylase from photosynthetic
organisms (cyanobacteria). Our results showed that Nostoc
could biotransform both MAs(V) and MAs(III) into As(III).
For MAs(V) demethylation by Nostoc, the detection of
MAs(III) at the early stage indicated that MAs(V)
demethylation was a two-step process: MAs(V) reduction
and MAs(III) demethylation. The previous research showed
that two microbes were required for MAs(V) reduction and
MAs(III) demethylation, respectively.29 Herein, our results
showed that Nostoc could carry out both steps. The tiny
amounts of intermediate MAs(III) suggested that NsArsI
demethylated MAs(III) to As(III) as soon as MAs(III) was
produced through MAs(V) reduction. Consequently, MAs(V)
reduction may be the rate-limiting step of MAs(V)
demethylation by Nostoc. However, the enzyme in Nostoc
responsible for MAs(V) reduction to MAs(III) is still not
known. For MAs(III) demethylation by Nostoc, As(III) as the
main product of MAs(III) demethylation was not extensively
accumulated within the Nostoc cells becauseAs(III) can be
extruded by efflux via Acr3 or ArsB.42 However, in Nostoc cells,
MAs(V) was detected as the main arsenic species after exposure
to MAs(III). Enzymatic MAs(III) oxidation may be one of the
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Environ. Sci. Technol. 2015, 49, 14350−14358
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Environmental Science & Technology
photosynthetic prokaryotic organisms may play a significant
role in As biogeochemical cycling.
of NsArsI, kinetic characterization of NsArsI suggested that
there are multiple ligand-binding sites in one protein molecule.
The turnover number for NsArsI was only 0.079 min−1, and the
possible reason for the low value is that both the enzyme and
the substrate for MAs(III) demethylation are readily oxidized.11
The C-terminal truncated derivatives of NsArsI with different
length of nonconserved residues deletion showed restricted
activity for C·As cleavage. Meanwhile, the C-terminal tail of
AkbC as a type I extradiol dioxygenase is responsible for
substrate specificity by blocking the entrance and plays a role in
shielding the binding pocket from oxidation and the correct
positioning of the substrate.52 Hence, we suggest that the Cterminal residues of NsArsI may contribute to the catalytic
activity by helping the substrate positioning for enzymatic
attack, although these residues are not directly involved in the
catalytic process. More studies are needed for the further
understanding of reaction mechanism of NsArsI by the
combined use of structural, kinetic, and computational
approaches. ArsI10 with the highest activity of the truncated
derivatives may be the first choice for use in crystallization to
determine the crystal structure.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.5b03357.
Figure S1: As species in the cell lysate of Nostoc exposed
to MAs(III) for the indicated times. Figure S2: Multiple
alignment of ArsI homologues. Figure S3: Graph of SDSPAGE for purified full-length NsArsI (ArsIall) and two
truncated derivatives (ArsI10 and ArsI23). Figure S4:
Elution profiles of NsArsI and molecular-mass standards
from a HiLoad 16/600 Superdex 75 column. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 86-592-6190977; fax: 86-592-6190977; e-mail:
[email protected].
■
Author Contributions
∥
ENVIRONMENTAL IMPLICATIONS
Methylated arsenicals such as MSMA are broad-spectrum
herbicides that are widely used in agriculture, and approximately 1 360 000 kg of MSMA have been applied annually in
the United States (www.epa.gov/oppsrrd1/REDs/organic_
arsenicals_red.pdf). Although the usage of MSMA is now
restricted to sod farms, golf courses, highway rights-of-way, and
cotton fields by the United States Environment Protection
Agency (USEPA),53 the repeated application of MSMA has led
to the contamination of soils.54 In addition, other organoarsenicals containing aromatic compounds such as roxarsone
(4-hydroxy-3-nitrophenylarsonic acid) have been extensively
used as growth promoters in intensive animal farming.
Although the USEPA has withdrawn approval of roxarsone in
animal feed formulations55 in light of environmental concerns
of roxarsone used in broiler poultry feed,56 it may still be widely
used in the animal industry in other parts of the world. The
microbial degradation of organoarsenicals will be an important
process in controlling the behavior and fate of organoarsenics in
the environment. Except for catalyzing MAs(III) demethylation, ArsI also has the ability to cleave the C·As bond in
trivalent roxarsone and other aromatic arsenicals.11 Cyanobacteria are abundant in the aquatic environments and soils. In our
study, Nostoc with NsarsI was shown to degrade methylated
arsenic into As(III), and it is very likely that Nostoc can degrade
aromatic arsenicals, too. Therefore, it is believed that
cyanobacteria are likely involved in organoarsenic degradation
and may play a critical role in arsenic biogeochemistry.
In conclusion, we have demonstrated that photosynthetic
organism Noctoc is capable of demethylation of MAs(III), and
we functionally characterized a novel C·As lyase, NsArsI, from
Nostoc. NsArsI exhibited the ability of C·As cleavage to
demethylate MAs(III). MAs(III) demethylation is an important
mechanism of MAs(III) detoxification in Nostoc. Cyanobacteria,
including Nostoc, are well-known for their contribution to
As(III) methylation14,35 and arsenosugars36 and arsenolipids57
biosynthesis. Our results of cyanobacteria involved in
methylarsenic demethylation open up new horizons in the As
biotransformation by cyanobacteria. Given the high abundance
of cyanobacteria in aquatic systems, wetlands, and soils, these
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Professor Wen-Li Chen of Huazhong Agricultural
University for providing Nostoc sp. PCC 7120. This work is
supported by the National Natural Science Foundation of
China (31270161), the Chinese Academy of Sciences
(XDB15020402), and the Natural Science Foundation of
Fujian Province (2014J01141).
■
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