AS3MT Human
Description
Introduction to AS3MT Human
AS3MT (Arsenic [+3 Oxidation State] Methyltransferase) is a human enzyme encoded by the AS3MT gene (10q24.32). It catalyzes the transfer of methyl groups from S-adenosylmethionine (SAM) to trivalent arsenicals, producing methylated metabolites such as monomethylarsonic acid (MAs[III/V]) and dimethylarsinic acid (DMAs[III/V]) . This enzyme plays a dual role: while methylation facilitates arsenic excretion, it also generates toxic and carcinogenic trivalent intermediates .
Key Features:
-
Catalytic Domains:
Table 1: Critical Cysteine Residues in hAS3MT
-
Substrate Preference:
Common Variants and Population Frequencies:
-
rs7085104: Associated with reduced dimethylarsenic (DMAs) excretion and increased schizophrenia risk .
-
rs11191439 (M287T): Alters methylation efficiency; linked to arsenic-related cancers .
Table 2: Select AS3MT Polymorphisms and Metabolic Effects
Metabolic Pathway:
Paradoxical Outcomes:
-
Detoxification: DMAs(V) excretion reduces systemic arsenic burden .
-
Toxicity: Trivalent intermediates (MAs(III)/DMAs(III)) are genotoxic and carcinogenic .
Table 3: Arsenic Metabolites in Humanized (Hs) vs. Wild-Type (WT) Mice
| Parameter | Hs Mice (Human AS3MT) | WT Mice (Mouse As3mt) |
|---|---|---|
| Urinary DMAs% | 35–40% | 70–80% |
| Tissue Retention | ↑↑ (Adrenal, liver) | ↓↓ |
| Methylation Efficiency | Low (Human-like) | High |
Tissue-Specific Expression
-
High Expression: Adrenal glands, liver, and parathyroid glands .
-
Neurological Relevance: Expressed in neurons and astrocytes; linked to epilepsy and schizophrenia .
Disease Associations
-
Cancer: Prolonged retention of trivalent metabolites increases bladder/lung cancer risk .
-
Neurological Disorders: Variants correlate with epilepsy susceptibility and schizophrenia .
-
Developmental Toxicity: Impaired methylation exacerbates arsenic-induced birth defects .
Research Advancements
-
Humanized Mouse Models: Hs mice mimic human arsenic metabolism, showing delayed excretion and tissue accumulation .
-
Inhibitor Development: Small molecules targeting hAS3MT reduce trivalent metabolite generation .
-
Gene Regulation: The AS3MT promoter contains a GC box bound by Sp1, influencing tissue-specific expression .
Product Specs
VINLVPDKQQ VLQEAYRVLK HGGELYFSDV YTSLELPEEI RTHKVLWGEC LGGALYWKEL AVLAQKIGFC PPRLVTANLI TIQNKELERV IGDCRFVSAT FRLFKHSKTG PTKRCQVIYN GGITGHEKEL MFDANFTFKE GEIVEVDEET AAILKNSRFA QDFLIRPIGE KLPTSGGCSA
LELKDIITDP FKLAEESDSM KSRCVPDAAG GCCGTKKSC.
Q&A
What is the function of human AS3MT and its role in arsenic metabolism?
Human AS3MT catalyzes the transfer of methyl groups from S-adenosylmethionine (SAM) to arsenite [As(III)], producing methylarsenicals including monomethylarsonic acid (MAs), dimethylarsinic acid (DMAs), and traces of trimethylarsenic compounds . This enzyme is crucial for arsenic biotransformation in humans, primarily occurring in the liver but also expressed in other tissues . The methylation process has a paradoxical nature - it both detoxifies arsenic and simultaneously transforms it into potentially carcinogenic species. AS3MT is a member of a large superfamily of methyltransferases involved in various physiological functions .
What structural elements are critical for human AS3MT function?
The human AS3MT enzyme contains seven cysteine residues, with four conserved cysteines (Cys32, Cys61, Cys156, and Cys206) and three non-conserved ones (Cys72, Cys85, and Cys250) . Site-directed mutagenesis studies demonstrate that all four conserved cysteines are essential for methylating As(III), while only Cys156 and Cys206 are required for methylating MAs(III) . Homology modeling based on a thermophilic orthologue indicates that Cys156 and Cys206 form the binding site for As(III), while Cys32 and Cys61 are positioned to form a disulfide bond during the catalytic cycle .
How is the human AS3MT gene expressed and regulated?
The human AS3MT gene shows tissue-specific expression patterns. It is most highly expressed in the adrenal gland, with lower expression levels in liver, brain, and peripheral blood mononuclear cells . Importantly, it is not expressed in human keratinocytes, urothelial cells, or brain microvascular endothelial cells, indicating tight transcriptional regulation . The core promoter region of human AS3MT contains a GC box to which the stress-related transcription factor Sp1 binds, suggesting involvement of stress response elements in regulating AS3MT expression .
What is the current understanding of the pathway for human AS3MT arsenic methylation?
The pathway of human AS3MT arsenic methylation appears to involve elements of both the Challenger pathway and the pathway proposed by Hayakawa et al. . Key observations include:
-
The preferred substrate appears to be the glutathione conjugate As(GS)₃ rather than inorganic As(III), as shown by faster binding in fluorescence assays .
-
The primary product of the first methylation round is trivalent MAs(III), not pentavalent MAs(V) .
-
The MAs(III) intermediate remains enzyme-bound until it undergoes a second methylation .
-
The process involves oxidation and reduction of arsenic species as enzyme-bound intermediates .
The methylation pathway produces more toxic and carcinogenic trivalent methylarsenicals, challenging earlier understandings of arsenic detoxification .
How do genetic polymorphisms in AS3MT affect arsenic metabolism capacity?
Genetic variations in AS3MT are significantly associated with differences in arsenic methylation capacity. SNP (Single Nucleotide Polymorphism) studies show that certain variants can alter the efficiency of converting inorganic arsenic to methylated forms . For instance, the M287T polymorphism affects enzyme catalytic properties. When comparing wild-type AS3MT with AS3MT/M287T using S-adenosylmethionine, arsenite, or methylarsonous acid (MAs III) as substrates and various endogenous reductants (GSH, thioredoxin, NADPH), differences in methylation efficiency become apparent . These genetic variations may explain population differences in susceptibility to arsenic toxicity and arsenic-related diseases.
What experimental systems are optimal for studying human AS3MT activity in vitro?
Highly active recombinant human AS3MT can be produced using codon-optimized synthetic genes expressed in E. coli . Key experimental considerations include:
-
Codon optimization for E. coli expression significantly improves translation, protein folding, and activity compared to cDNA-expressed enzyme .
-
The purified enzyme requires endogenous reductants for activity, including thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH) .
-
Alternative reducing agents such as Tris(2-carboxyethyl)phosphine (TCEP) can also support AS3MT activity .
-
Single-tryptophan derivatives of AS3MT can be used for fluorescence-based binding assays to study substrate interactions .
-
Catalytic assays should measure both trivalent and pentavalent arsenical species, as the trivalent forms are unstable and require careful handling .
What is the role of cysteines in AS3MT and how can they be studied?
The four conserved cysteine residues in human AS3MT (Cys32, Cys61, Cys156, and Cys206) have distinct functional roles that can be studied through site-directed mutagenesis :
| Cysteine Residue | Role in AS3MT | Effect of Mutation to Serine |
|---|---|---|
| Cys32 | Forms disulfide bond with Cys61 | Cannot methylate As(III) but retains MAs(III) methylation |
| Cys61 | Forms disulfide bond with Cys32 | Cannot methylate As(III) but retains MAs(III) methylation |
| Cys156 | Forms As(III) binding site | Cannot methylate any arsenical species |
| Cys206 | Forms As(III) binding site | Cannot methylate any arsenical species |
| Cys72, Cys85, Cys250 | Non-conserved, non-critical | Retain methylation activity when mutated |
These findings suggest that Cys156 and Cys206 are directly involved in arsenic binding at all steps of the pathway, while Cys32 and Cys61 play a regulatory role through disulfide bond formation .
How can molecular modeling enhance our understanding of AS3MT function?
Homology modeling provides valuable insights into human AS3MT structure and function . A homology model of hAS3MT can be built on the structure of PhAs(III)-bound CmArsM (a thermophilic orthologue) using automated servers such as SWISS-MODEL . The quality of such models can be estimated using scoring functions like QMEAN . In silico docking with substrates such as SAM can be performed using tools like PATCHDOCK .
These models reveal that:
-
Cys156 and Cys206 form the binding site for arsenicals
-
Cys32 and Cys61 are positioned to form a disulfide bond
-
The SAM binding site is positioned to facilitate methyl transfer to As(III)
Such molecular models can guide hypothesis formation and experimental design for further functional studies.
What techniques are available for studying AS3MT gene expression regulation?
Several approaches can be used to study the transcriptional regulation of AS3MT :
-
Reporter gene constructs containing various fragments of the AS3MT promoter region can be created to identify regulatory elements .
-
The core promoter region of human AS3MT (positions -833 to +630 relative to the transcription start site) can be synthesized and cloned into appropriate vectors .
-
Various fragments of the promoter can be obtained by PCR and tested for activity in different cell types .
-
Binding of transcription factors like Sp1 to regulatory elements such as the GC box can be studied using techniques like chromatin immunoprecipitation .
-
RNA-seq analysis can be employed to compare AS3MT expression across different tissues and under various conditions .
These methods help elucidate the tissue-specific expression patterns observed for AS3MT and the regulatory mechanisms involved.
How does AS3MT expression affect susceptibility to arsenic toxicity?
Clonal human urothelial cells expressing rat AS3MT show increased susceptibility to acute toxicity from arsenite exposure . This suggests that AS3MT expression and the resulting methylation activity can modulate cellular responses to arsenic compounds. The paradoxical role of AS3MT in both detoxification and bioactivation of arsenic has important implications for understanding differential susceptibility to arsenic toxicity among individuals and populations with varying AS3MT expression or polymorphisms . Transcriptional profiling of cells expressing AS3MT reveals altered gene expression patterns in response to arsenic exposure, which may contribute to the observed differences in susceptibility .
What are the implications of AS3MT-mediated arsenic metabolism for carcinogenesis?
AS3MT-mediated arsenic biomethylation generates both pentavalent species (MAs(V), DMAs(V), TMAs(V)O) and more toxic trivalent methylarsenicals (MAs(III), DMAs(III), TMAs(III)) . The trivalent methylated species are particularly concerning as they demonstrate higher cytotoxicity and genotoxicity than inorganic arsenic . The conversion of inorganic arsenicals into carcinogenic trivalent methylated species represents a biotransformation of a small molecule into a carcinogen, a process that parallels other known xenobiotic activations . Understanding the balance between detoxification and activation pathways is crucial for assessing cancer risk from arsenic exposure in populations with different AS3MT genetic backgrounds.
Product Science Overview
Arsenic methyltransferase, also known as arsenite methyltransferase (AS3MT), is an enzyme that plays a crucial role in the biomethylation of arsenic. This enzyme is responsible for the conversion of inorganic arsenic into its methylated forms, which are generally less toxic and more easily excreted from the body. The human recombinant form of this enzyme is often used in research to study its structure, function, and role in arsenic metabolism.
The human AS3MT gene encodes a protein consisting of 375 amino acids with a molecular weight of approximately 41.7 kDa . This enzyme contains several cysteine residues that are essential for its catalytic activity. The cysteine residues form disulfide bonds, which are crucial for the enzyme’s function. For instance, the disulfide bond between Cys250 and Cys32 is reduced by glutathione (GSH) or other reductants before the enzymatic methylation of arsenite .
AS3MT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to arsenic, producing monomethyl and dimethyl arsenicals . The enzyme’s active site binds to arsenite (iAs^3+) and facilitates its methylation. The methylation process involves the formation of intermediate complexes, such as Cys250-S-adenosylhomocysteine (AdoHcy), which suggests that Cys250 is involved in the transmethylation process .
Arsenic is a ubiquitous environmental contaminant that poses significant health risks. The methylation of arsenic by AS3MT is a critical detoxification pathway that reduces the toxicity of arsenic and enhances its excretion. The methylated forms of arsenic, such as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), are less toxic and more water-soluble, facilitating their elimination from the body.
Recombinant human AS3MT is widely used in research to understand the molecular mechanisms of arsenic methylation and its implications for human health. Studies have shown that mutations in the AS3MT gene can affect the enzyme’s activity and influence an individual’s susceptibility to arsenic toxicity . Additionally, research on AS3MT has provided insights into the role of glutathione and other reductants in the methylation process .
