Pathway of Human AS3MT Arsenic Methylation

Overview

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Key numbers

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• range of acute and chronic toxic effects that increase the MAs(V).15,16 However, with careful handling, trivalent
• TMAs.10 Human AS3MT is primarily a liver enzyme that is a unstable MAs(III) and DMAs(III), which rapidly oxidize
• methylation pathway and that Cys32 and Cys61 play a different (Millipore, Billerica, MA). Glycerol was added to 20% (v/v), and the
• closely enough to form a disulfide bond. We propose that this phase (A600nm ∼ 0.5−0.6) before induction with 0.2% (w/v) L-
• reactions were terminated by adding 10% (v/v) H2O2 to oxidize all whether either construct could attain steady state, the most
• position (Table S2), creating single-tryptophan derivatives Trp73 and enterokinase sites, and eight histidines at the C-terminus
• For steady-state measurements, both emission and excitation tryptophan residues at each position (Table S2), creating
• estimated on the basis of the QMEAN scoring function.32 The model as that produced by As(GS)3 in 1 s, but the quenching by
• fluorescence of Trp73 hAS3MT was assayed at 25 °C in degassed (right), DMAs. The data are the mean ± SE (n = 3).
• withdrawn at the indicated times, and the reaction terminated by addition of 10% (v/v) H2O2, final concentration, and analyzed by HPLC-ICP-MS.
• period was determined using GSH and the Trx system (Figure with 80% conversion with the synthetic enzyme. Thus, the
• 15% of the total arsenic in the reaction mixture. The production inference is that they differ in conformation, perhaps because
• until 97% of the total arsenic was present as DMAs. These enzyme, which was optimized for E. coli expression. One
• major bound arsenical form released from the enzyme. A small the acceptable range.32 The homology model incorporated 308
• similar amount of MAs(III) was nonenzymatically oxidized to arrangement of hAS3MT was nearly the same as that of the
• nonconserved cysteines (Cys72, Cys85 Cys226, Cys250, provides testable predictions. The biochemical data demon-
• addition of 10% (v/v) H2O2, final concentration. Protein was removed before analysis, and soluble arsenicals were analyzed by reversed-phase
• and the products were pentavalent.1 A more recent proposal by As(GS)3 complex is unstable in vitro, so it is challenging to

Methods (brief)

• MAs(III), DMAs(III), and TMAs(III) are intermediates but
• exposure.3−6 In humans, arsenic is associated with a broad humans primarily excrete DMAs(V) and to a lesser extent
• In humans, S-adenosylmethionine As(III) methyltransferase MAs(GS)2 and DMAs(GS).2 In this pathway, there is no
• from SAM to As(III), producing MAs, DMAs, and traces of throughout the catalytic cycle. The conjugates dissociate to
• TMAs.10 Human AS3MT is primarily a liver enzyme that is a unstable MAs(III) and DMAs(III), which rapidly oxidize
• member of a large superfamily of methyltransferases that are nonenzymatically in air to MAs(V) and DMAs(V), the primary
• trivalent MAs(III) and DMAs(III).2,10,13 genes were subsequently cloned.20−22 The enzyme methylates
• dimethylarsenate (DMAs(V)) and a lesser amount of Published: October 17, 2014
• producing both MAs(III) and DMAs(III), with either Trx or synthetic hAS3MT gene was cloned as an NcoI/SalI digest from
• DMAs(V), but, like the other hAS3MT expressed from a CCAGCCATGGCTGCACTTCGTGACGCTGAGA-3′ (NcoI site
• As(III) to DMAs(V), so this may also represent only a single was cloned into pBAD-Myc/His-A as an NcoI/SalI digest, generating
• pressure liquid chromatography (HPLC) (PerkinElmer Series 2000) models.35,36
• plasma mass spectrometry (ICP-MS) using an ELAN DRC-e human AS3MT was expressed from a cDNA clone and purified
• monochromator slits were set at 1 nm. Samples were excited at 295 single-tryptophan derivatives Trp73 (W203L/W213Y), Trp203
• methylated to both MAs and DMAs, but the extent of
• methylation and the ratio of DMAs/MAs varied (Figure 2). In
• to oxidize all arsenic species, which were speciated by HPLC-ICP-MS.
• fluorescence of Trp73 hAS3MT was assayed at 25 °C in degassed (right), DMAs. The data are the mean ± SE (n = 3).

Implications

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Wiki pages this source may touch

• Fish — marine, non-predatory (sardines, anchovies, salmon, cod)
• Arsenic

Verification notes

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Update history

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