High-throughput screening-compatible assays of As(III) S-adenosylmethionine methyltransferase activity

Abstract

Arsenic is a naturally existing toxin and carcinogen. As(III) S-adenosylmethionine methyltransferases (AS3MT in mammals and ArsM in microbes) methylate As(III) three times in consecutive steps and play a central role in arsenic metabolism from bacteria to humans. Current assays for arsenic methylation are slow, laborious, and expensive. Here we report the development of two in vitro assays for AS3MT activity that are rapid, sensitive, convenient, and relatively inexpensive and can be adapted for high-throughput assays. The first assay measures As(III) binding by the quenching of the protein fluorescence of a single-tryptophan derivative of an AS3MT ortholog. The second assay utilizes time-resolved fluorescence resonance energy transfer to directly measure the conversion of the AS3MT substrate, S-adenosylmethionine, to S-adenosylhomocysteine catalyzed by AS3MT. These two assays are complementary, one measuring substrate binding and the other catalysis, making them useful tools for functional studies and future development of drugs to prevent arsenic-related diseases.

The Arsenic Methylation Cycle: How Microbial Communities Adapted Methylarsenicals for Use as Weapons in the Continuing War for Dominance

“…Bacteria shield themselves from AST toxicity by expression of arsN. first two steps of methylation, As(III) to MAs(III) and MAs(III) to DMAs (the presumed product, DMAs(III) were determined (Dong et al, 2015). In the presence of GSH, the K m for wild-type human AS3MT enzymes was measured as approximately 1.6 µM for As(III) and 0.7-0.8 µM for MAs(III).…”

Arsenic Biotransformations in Microbes and Humans, and Catalytic Properties of Human AS3MT Variants

“…We showed previously that the product of the synthetic gene can be used in catalytic amounts with the Trx/TR/GSH assay. A large excess of substrate over enzyme ensures that there are multiple rounds of methylation during the assay time, which, with the TR-FRET assay, is linear up to 5 min (Dong et al 2015b). In addition, the TR-FRET assay allows the two methylation reactions (iAs→MAs and MAs→DMAs) to be determined independently, an accomplishment not possible in previous studies.…”

“…MAs(V) was reduced to trivalent MAs(III) using Na 2 S 2 O 3, Na 2 S 2 O 5 , and H 2 SO 4 , and adjusted to pH 6.5 with NaOH, as described . The identity of the reduction products were confirmed by high performance liquid chromatography (HPLC) coupled to inductively coupled mass spectroscopy (ICP-MS), as described (Dong et al 2015b). The substrates of for methylation were the glutathione (GSH) conjugates As(GS) 3 and MAs(GS) 2, which were prepared by incubation of 1 mM As(III) or MAs(III) with a 4-fold molar excess of GSH for 5 h at 23 °C in degassed buffers under argon, as described .…”

“…The TR-FRET assay measures formation of SAH from SAM and thus cannot be used for determination of SAM kinetics (Dong et al 2015b), so the kinetics for SAM as substrate were determined by formation of DMAs from MAs(III) as a function of SAM concentration using HPLC-ICP-MS. This measures only the second methylation step but was used because the C61S variant cannot methylate As(III).…”

“…hAS3MT activity was assayed with two different procedures. The time-resolved Förster resonance energy transfer (TR-FRET) assay measures conversion of SAM to Sadenosylhomocysteine (SAH) at short times using an EPIgeneous Methyltransferase Assay kit (Cisbio Bioassays, Bedford, MA)(Dong et al 2015b). The assay was carried out using a low volume 384-well microtiter plate in a buffer consisting of 50 mM NaH 2 PO 4 , 0.3 M NaCl, 1 μM purified hAS3MT, 0.5 mM GSH, 1 μM Trx, 0.3 μM TR and 0.03 mM NADPH and 10 μM of either As(GS)3 or MAs(GS) 2 , unless otherwise indicated.The plates were incubated at 37 °C for 2 min with shaking in an Eppendorf ThermoMixer C before adding SAM at 10 μM, final concentration (unless otherwise indicated), to initiate the reaction.…”