FEMS Microbiology Letters 235 (2004) 95–99
www.fems-microbiology.org
An origin for arsenobetaine involving bacterial formation of
an arsenic–carbon bond
Alisdair W. Ritchie a, John S. Edmonds b, Walter Goessler c, Richard O. Jenkins
a,*
a
b
Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK
Endocrine Disrupter Research Laboratory, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
c
Institute for Chemsitry – Analytical Chemistry, Karl Franzens Universit€at Graz, Universit€atsplatz 1, 8010 Graz, Austria
First published online 22 April 2004
Abstract
Lysed-cell extract of a Pseudomonas sp. was shown to catalyse bioconversion of dimethylarsinoylacetate to arsenobetaine and
dimethylarsinate. Provision of the universal methyl donor S-adenosylmethionine to bioconversion mixtures promoted both the rate
and extent of arsenobetaine formation. These findings suggest that in the proposed biosynthesis of arsenobetaine from dimethylarsinoylethanol, oxidation (i.e. the formation of the carboxymethyl group of dimethylarsinoylacetate) would precede the reduction
and methylation at the arsenic atom. The presence of enzyme(s) capable of methylating dimethylarsinoylacetate in a bacterial isolate
from marine mussel (Mylitus edulis), highlights a possible direct involvement of prokaryotic organisms in the biosynthesis of organoarsenic compounds within marine animals.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Arsenobetaine; Dimethylarsinoylacetate; Pseudomonas; S-adenosylmethionine; Arsenic; Dimethylarsinate
1. Introduction
It has been known for some years that marine animals naturally contain appreciable quantities of arsenic
and that, in most cases, this arsenic is present almost
entirely as trimethylarsonioacetate (arsenobetaine;
Fig. 1, 5) [1,2]. Arsenobetaine is metabolically stable [3]
– it is rapidly excreted through the human kidney, and
presents no toxic hazard. Until relatively recently it was
thought that the natural occurrence of arsenobetaine
was restricted to the marine environment but it has now
been shown to occur in mushrooms [4] and in earthworms [5] living in arsenic polluted environments.
Although arsenobetaine was discovered as early as
1977 [6] and is widespread in marine biota, information
about its biosynthesis is lacking. It is speculated that
arsenobetaine is formed via degradation of arsenosu*
Corresponding author. Tel.: +44-116-2506306; fax: +44-1162577287.
E-mail address: [email protected] (R.O. Jenkins).
gars. The overwhelming bulk of algal arsenosugars are
dimethylarsinoylribosides (Fig. 1, 1) and thus contain
two methyl groups and a 5-deoxyriboside group attached to the arsenic atom. The biosynthesis of the
dimethylarsinoylribosides has been previously considered at length [7]. It is very likely that S-adenosylmethionine (SAM) is the source of both the methyl groups
and the ribose moiety attached to the arsenic atom.
Probably the two-carbon (carboxymethyl) side-chain in
arsenobetaine is derived from breakdown of the ribosecontaining portion of the dimethylarsinoylribosides.
Anaerobic microbial activity derived from marine sand
very quickly converts algal dimethylarsinoylribosides to
dimethylarsinoylethanol (Fig. 1, 2) which is readily seen
as a precursor of arsenobetaine. Curiously, attempts to
bring about the further reduction and methylation of the
arsenic atom of this compound by the sort of unspecified
microbial activity that facilitated its formation have
been unsuccessful [8]. In addition, fish when supplied
with either dimethyarsinoylethanol or dimethylarsinoyl
acetic acid (Fig. 1, 4) in their food also failed to
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.04.016
Downloaded from http://femsle.oxfordjournals.org/ by guest on May 16, 2016
Received 3 February 2004; received in revised form 6 April 2004; accepted 7 April 2004
96
A.W. Ritchie et al. / FEMS Microbiology Letters 235 (2004) 95–99
O
Me2As
O
OR
O
Me2AsCH2CH2OH
2. Dimethylarsinoylethanol
OH OH
1. Dimethylarsinoylribosides ;
R = -CH2CHOHCH2OH
Oxidation
Reduction and
methylation
O
+
Me3As CH2CH2OH
3. Arsenocholine ion
Me2AsCH2COOH
4. Dimethylarsinoyl acetic acid
Oxidation
+
-
Me3As CH2COO
5.
Arsenobetaine
Fig. 1. Scheme for the biosynthesis of arsenobetaine from dimethylarsinoylribosides.
accumulate them as arsenocholine (Fig. 1, 3) or arsenobetaine [9].
There have been, however, several studies of the microbial degradation of arsenobetaine [10,11]; we have
recently reported on two bacterial isolates from marine
mussel (Mylitus edulis) capable of degrading arsenobetaine to dimethylarsinate via dimethylarsinoylacetate
[12]. In the present work we report for the first time the
biosynthesis of arsenobetaine from dimethylarsinoylacetate by a lysed-cell extract of one of these isolates.
2. Materials and methods
2.1. Materials and microorganisms
Arsenobetaine was prepared using the procedure described by Lagarde et al. [13]. 2-Dimethylarsinoyl acetic
acid was prepared according to the method of Wigren
[14,15]. Sodium dimethylarsinate (Me2 As(O)O Naþ )
was obtained from Sigma–Aldrich. Pseudomonas fluorescens A NCIMB 13944, previously described as
degrading arsenobetaine to dimethylarsinate via dimethylarsinoylacetate in pure culture [12], is deposited with
the National Culture of Industrial and Marine Bacteria,
Aberdeen, UK.
2.2. Culture and preparation of lysed-cell extracts
Pseudomonas sp. NCIMB 13944 was grown in 6 1 l
flasks containing 200 ml minimal salts medium supple-
mented with sodium succinate (2 g l1 ), in the absence of
arsenic compounds, at 25 °C on a rotary shaker (150
rpm) for 3 days. The minimal salts medium contained
(g l1 ): KH2 PO4 (2); NH4 Cl (3); MgSO4 7H2 O (0.4);
trace element solution (2 ml) [16]; NaCl (30), final pH,
6.8 adjusted with KOH. The trace element solution
comprised (g l1 ): ethylenediaminetetraacetic acid (50);
ZnSO4 7H2 O (22); MnCl2 4H2 O (5.54); FeSO4 7H2 O
(4.99); (NH4 )6 Mo7 O2 4H2 O (1.1); CuSO4 5H2 O
(1.57); CoCl2 6H2 O (1.61), final pH, 7.2. Cells were
harvested by centrifugation at 6000g for 30 min at 4 °C,
washed twice in 50 mM potassium phosphate buffer (pH
7.2), and resuspended in 30 ml of the buffer. Cells were
disrupted using a MSE Soniprep 150 ultrasonic disintegrator (MSE Scientific Instruments, Crawley, UK), at
an amplitude of 10–12 lm for 20 30 s, with cooling on
ice between cycles. Debris was removed by centrifugation at 100,000g for 60 min; the supernatant was decanted and termed the lysed-cell extract.
Protein concentration was determined by the Bradford method, using Biorad (Hercules) protein assay reagent and bovine serum albumin (Sigma–Aldrich) as
standards (0–0.2 mg protein ml1 ). The protein concentration of the lysed-cell extract was 2.0 mg ml1 .
2.3. In vitro assay for arsenobetaine biosynthesis
Lysed-cell extract (0.1 ml) was incubated at 30 °C for
up to 300 min in 0.9 ml of 50 mM potassium phosphate
(pH 7.2) buffer. All incubations contained a final concentration of 1 mg As l1 as dimethylarsinoylacetate;
Downloaded from http://femsle.oxfordjournals.org/ by guest on May 16, 2016
Reduction and
methylation
(this study)
A.W. Ritchie et al. / FEMS Microbiology Letters 235 (2004) 95–99
SAM was added to some assay mixtures to a final
concentration of 1 mg l1 . Aliquots (0.1 ml) of assay
mixtures were taken at intervals, diluted 10-fold in water
and frozen at )20 °C ready for analysis. The arsenic
compounds were determined by high-performance liquid chromatography/inductively coupled plasma mass
spectrometry (HPLC/ICPMS).
2.4. HPLC/ICPMS analysis
Intensity
DMAA
DMA
AB
t = 0 min
t = 60 min
t = 120 min
3. Results
Bioconversion of dimethylarsinoylacetate by a lysedcell extract of the P. fluorescens A strain, both in the
presence and absence of added SAM (supplied as potential methyl donor), is shown in Fig. 2. In both types of
incubations, arsenobetaine and dimethylarsinate were
formed as sole detectable arsenic containing products of
dimethylarsinoylacetate bioconversion; recovery of arsenic in these products was P 98% of that supplied in the
form of dimethylarsinoylacetate. Confirmation of the
formation of arsenobetaine from dimethylarsinoylacetate by the lysed-cell extract was confirmed by HPLC/
ICPMS analysis on a cation-exchange column (Fig. 3).
Co-injection of assay incubation mixtures and
(organo)arsenic standards (arsenobetaine, dimethylarsinoylacetate, dimethylarsinate, methylarsinate, trimethylarsine oxide, arsenocholine, dimethylarsinoylethanol,
tetramethylarsonium ion, arsenate and arsenite), with
subsequent chromatographic separations on both anionand cation-exchange columns, was also used to
confirmed the formation of arsenobetaine from dimethylarsinoylacetate by the lysed-cell extract.
Supply of SAM promoted both the rate and extent of
arsenobetaine formation (Figs. 2 and 4). In the presence
of SAM, the maximum specific rate of arsenobetaine
formation was 6.0 lg As min1 (mg protein)1 (67%
higher than in absence of SAM), with 134 lg of arsenic
in the form of arsenobetaine after 180 minutes incubation (almost 4-fold higher than in absence of SAM).
SAM however did not influence the maximum specific
t = 180 min
(a)
DMAA
AB
Intensity
Intensity
DMAA
DMA
DMA
AB
t = 0 min
t = 0 min
t = 60 min
t = 60 min
t = 120 min
t = 120 min
t = 180 min
t = 300 min
(b)
0
1
2
3
4
t = 180 min
5
Time (min)
0
2
1
3
Time (min)
Fig. 2. (a,b) Bioconversion of dimethylarsinoylacetate by lysed-cell
extract of P. fluorescence A NCIMB 13944. Chromatograms obtained
by HPLC/ICPMS, with separation of arsenic species on a Hamilton
PRP-X100 anion exchange column. Arsenic species in assay mixtures
at intervals up to 300 min incubation; all assay mixtures were supplemented with dimethylarsinoylacetate (DMAA) to 1 mg As l1 immediately prior to incubation, and diluted 10-fold prior to analysis. (a)
Assay mixtures not supplemented with S-adenosyl methionine and (b)
supplemented with S-adenosylmethionine to 100 lg l1 . Retention
times (min) of organoarsenic standards: arsenobetaine, AB (1.53);
dimethylarsinate, DMA (2.15); dimethylarsenoylacetate, DMAA
(3.22).
Fig. 3. Confirmation of the biotransformation of dimethyl arsinoylacetate to arsenobetaine by lysed-cell extract of P. fluorescence A
NCIMB 13944. Chromatograms obtained by HPLC/ICPMS, with
separation of arsenic species on a Zorbax 300-SCX cation-exchange
column. Arsenic species in assay mixtures at intervals up to 180 min
incubation; all assay mixtures were supplemented with dimethylarsinoylacetate (DMAA) to 1 mg As l1 immediately prior to incubation,
and diluted 10-fold prior to analysis. Assay mixtures were not supplemented with S-adenosylmethionine. Retention times (min) of organoarsenic standards: arsenobetaine, AB (1.94); dimethylarsinate,
DMA (1.40); dimethylarsenoylacetate, DMAA (1.69).
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An integrated Hewlett Packard HPLC/ICPMS
system, fitted with a Babbington nebuliser was used.
Anion-exchange chromatography was on a Hamilton
PRP-X100 column (250 mm 4.6 mm) at 30 °C, using
20 mM NH4 H2 PO4 as mobile phase (adjusted to pH 6.0
with 25% NH3 ðaqÞ ). Cation-exchange chromatography
was on a Zorbax 300-SCX column (150 mm 4.6 mm)
at 30 °C, using 20 mM aqueous pyridine as mobile phase
(adjusted to pH 2.6 with formic acid). For both columns, flow rate was 1.5 ml min1 and sample injection
volume was 20 ll.
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A.W. Ritchie et al. / FEMS Microbiology Letters 235 (2004) 95–99
rate of dimethylarsinate formation (3.7 lg As min1 (mg
protein)1 ), and after 180 minutes incubation had
increased the amount of arsenic in this bioconversion
product by only 23% to 94 lg As. After 300 minutes
incubation in the presence of SAM, 17 and 15% of
supplied arsenic (as dimethylarsinoylacetate) was recovered as arsenobetaine and dimethylarsinate respectively (Fig. 2).
4. Discussion
Chemical synthesis of arsenobetaine involves nucleophilic attack of the trivalent arsenic atom of trimethylarsine on the bromine-bearing carbon of ethyl
bromoacetate [3]. Methylation of arsenic (e.g. by the
bread mould Scopulariopsis brevicaulis – as in the
‘Challenger’ pathway [17]) follows a process that parallels the chemical synthesis, i.e. nucleophilic attack by a
reduced (electron-rich) arsenic species on a positively
charged carbon (the methyl-bearing carbon of SAM).
However, the natural transfer of a carboxymethyl group
to arsenic (necessary for the biosynthesis of arsenobetaine) by an analogous route is mechanistically unlikely.
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Fig. 4. (a,b) Influence of S-adenosylmethionine (SAM) on bioconversion of dimethylarsinoylacetate by lysed-cell extract of P. fluorescence
A NCIMB 13944. Assay mixtures were supplemented with dimethylarsinoylacetate (DMAA) to 1 mg As l1 immediately prior to incubation. (a) Arsenobetaine formation with ðÞ and without ðjÞ
addition of SAM. (b) Dimethylarsenic acid formation with ðÞ and
without ðjÞ addition of SAM. When supplied, SAM was added to
incubation mixtures to 100 lg l1 . Standard deviations (shown) are
based on three replicates.
The presence of arsenosugars in the early stages of
marine food chains and their occurrence with arsenobetaine in some animals suggested that arsenosugars
might be precursors of arsenobetaine in a biogenerative
process. Considerable breakdown of that part of the
molecule containing the sugar residue would have to
occur. Ideally such a degradative process should leave a
two-carbon chain attached to arsenic together with the
two methyl groups and oxygen already present. A reduction followed by an additional methylation, together
with oxidation of the new two-carbon side chain to form
a carboxymethyl group will yield arsenobetaine. Considerable experimental support for such a scheme was
provided by a non-specific anaerobic microbial treatment of marine algal fragments; the arsenosugars they
contained were quantitatively converted to dimethylarsinoylethanol, a compound that could very easily be
seen as a precursor of arsenobetaine in the terms just
outlined [18]. In particular, the ribosides attached to
arsenic had been degraded to the required simple twocarbon unit. Thus it seemed feasible that all groups attached to arsenic in arsenobetaine (the three methyl and
the carboxymethyl) were derived from a single source
(SAM) and were transferred to arsenic by a single process, namely, nucleophilic attack by reduced arsenic on a
positively charged carbon.
The demonstration of the microbial breakdown of
dimethylarsinoylribosides to yield the necessary 2-carbon chain only provides a plausible biosynthetic route
to arsenobetaine if the final reduction and methylation
of the arsenic atom is possible. Although mechanistically there would seem to be no impediment to such a
reaction as it parallels any single stage in the ‘Challenger’ pathway (reduction of arsenic followed by oxidative methylation) demonstration of its occurrence has
not been achieved until now.
Additional support, in particular for the degradative
aspects of this biosynthetic scheme for arsenobetaine,
has recently been provided by a chromatographic and
mass spectrometric study of the wide range of arsenic
compounds present in the kidney of the giant clam
Tridacna derasa [19]. In this case both arsenosugars and
arsenobetaine were present, and several compounds
could easily be seen as intermediates. It appeared that in
the clam kidney the aglycone chains of the arsenosugars
were progressively degraded by oxidation and decarboxylation, the ribose ring was then opened and the
resulting sugar alcohol chain degraded in the same
manner. The penultimate product was dimethylarsinoylacetate, which could be converted to arsenobetaine
by reduction and methylation at the arsenic atom.
Earlier work [18] left it unclear whether in the conversion of dimethylarsinoylethanol to arsenobetaine, reduction and methylation at the arsenic atom preceded or
followed the oxidation of the terminal carbon in the
two-carbon side chain. The work reported here has
A.W. Ritchie et al. / FEMS Microbiology Letters 235 (2004) 95–99
Acknowledgements
The Engineering and Physical Sciences Research
Council of the UK supported this work.
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indicated that the oxidation (i.e. the formation of the
carboxymethyl group) precedes the reduction and
methylation at the arsenic atom.
We have recently reported on the capability of P.
fluorescens A NCIMB 13944, a bacterium isolated from
Mytilus edulis (marine mussel), to degrade arsenobetaine
in whole cell bioconversions by initial cleavage of a
methyl-arsenic bond to form dimethylarsinoylacetate,
with subsequent cleavage of the carboxymethyl-arsenic
bond to yield dimethylarsinate [12]. During this study on
bacterial degradation of organoarsenic compounds, we
noted that growth of P. fluorescens A NCIMB 13944 in
the presence of dimethylarsinoylacetate resulted in 94%
removal of the organoarsenical from culture supernatants with appearance of some dimethylarsinate and arsenobetaine (each ca. 1–2% of the As supplied) (data not
shown). This stimulated us to investigate the possibility
of arsenobetaine formation by a prokaryotic organism,
using lysed-cell extract of the P. fluorescens A strain.
In the present study, we have found that a lysed-cell
extract of this organism converts dimethylarsinoylacetate not only to dimethylarsinate but also to arsenobetaine. We have also demonstrated that SAM markedly
promotes the bioconversion of dimethylarsinoylacetate
to arsenobetaine, which suggests that the bioconversion
is catalysed by a methyltransferase enzyme using SAM as
methyl donor. We have suggested previously [12] that
bioconversion of arsenobetaine to dimethylarsinoylacetate may be catalysed through the reversible action of a
methyltransferase, and the data reported here support
that view. Although bioconversion of dimethylarsinoylacetate by the P. fluorescens A strain does not require
induction of substrate, preincubation of cells with inorganic arsenic or organoarsenic compound(s) could
greatly enhance the specific activity of the enzymes involved.
As discussed above, two routes of biological formation of arsenobetaine from dimethylarsinoylethanol
have been proposed: one involving arsenocholine the
other involving dimethylarsinoylacetate as intermediate
[18]. The significance of the work reported here is that it
provides evidence for enzymatic methylation of dimethylarsinoylacetate, a key step in arsenobetaine biosynthesis via one of these routes. The presence of such
enzymatic activity in a bacterial species, isolated from
marine mussel, indicates a possible direct involvement of
prokaryotic organisms in biosynthesis of organoarsenic
compounds within marine animals.
99