title: 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.
authors: Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA.
authors: Hui
authors: H
authors: Dong
authors: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors: Wenzhong
authors: W
authors: Xu
authors: Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA.
authors: Jitesh K
authors: JK
authors: Pillai
authors: Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA.
authors: Charles
authors: C
authors: Packianathan
authors: Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA. Electronic address: brosen@fiu.edu.
authors: Barry P
authors: BP
authors: Rosen
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000032:analysis / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*
chemical_list: D012436:S-Adenosylmethionine; D008780:Methyltransferases
keywords: AS3MT; Arsenic; High-throughput screens; Methylation