bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.10.12.511968

METTL7A and METTL7B are responsible for S-thiol methyl transferase activity in liver

Russell, D. A.·Chau, M. K.·Shi, Y.·Maldonato, B. J.·Totah, R. A.
View original source ↗Export metadata ↓

Abstract

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMTs role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro--methylbenzylamine (DCMB), has no effect on the activity of METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, also acts as a thiolmethyltransferase. METTL7A exhibits similar biochemical properties to TMT, including inhibition by DCMB (IC50 1.2 {micro}M). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A1 and TMT1B1, respectively, that are responsible for TMT activity in liver microsomes. Significance StatementWe identified METTL7A (TMT1A1) and METTL7B (TMT1B1) as the enzymes responsible for the microsomal alkyl thiol methyltransferase activity. These are the first two enzymes directly associated with microsomal TMT activity. S-Methylation of many commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible, will improve our understanding of the DMPK properties of alkyl- or phenolic-thiol-containing therapeutics. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/511968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2419f9org.highwire.dtl.DTLVardef@254c5borg.highwire.dtl.DTLVardef@b81378org.highwire.dtl.DTLVardef@1064c79_HPS_FORMAT_FIGEXP M_FIG C_FIG

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Russell, D. A., Chau, M. K., Shi, Y., Maldonato, B. J., Totah, R. A.. 2022-10-13. METTL7A and METTL7B are responsible for S-thiol methyl transferase activity in liver. https://doi.org/10.1101/2022.10.12.511968

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Direct Bioconjugations of Diverse Payloads using an Extraordinarily Donor-Promiscuous Flavin Transferase

Site-specific protein conjugation is a widely used tool in chemical biology research and is the foundation of many biotechnological and biomedical applications. A key enabling technology are repurposed post-translational protein modifying enzymes, where variants with broad donor and acceptor substrate promiscuity were developed into general-purpose bioconjugation platforms. However, direct enzymatic attachment of bulky or structurally diverse payloads is often limited by low reaction efficiencies and difficulties in preparing suitable donors. Consequently, conjugation typically relies on two-step strategies in which an enzyme first installs a bioorthogonal handle on the target protein, followed by chemical attachment of the payload. These enzyme-first, chemistry-second approaches limit chemistry to bioorthogonal reactions that leave a molecular footprint. Here, we introduce direct ADDing, an enzymatic bioconjugation platform based on our discovery of the exceptional donor promiscuity of the flavin transferase ApbE. We show that ApbE accepts a remarkably broad range of donor substrates provided they carry an adenosine diphosphate (ADP) anchor. The prevalence of ADP-containing compounds in nature provides access to diverse building blocks and an extensive biochemical toolbox for donor generation. Exploiting these resources, we demonstrate multiple strategies for preparing compatible donors and achieve direct, site-specific enzymatic conjugation of diverse small molecules, peptides, proteins, and synthetic polymers.

biochemistry↗
bioRxiv2026

A respiratory microdomain links ubiquinone biosynthesis to succinate dehydrogenase in an apicomplexan mitochondrion lacking Complex I

Mitochondrial complex I (CI) is a major entry point for NADH-derived electrons and an important site of redox regulation in most eukaryotes. However, apicomplexan parasites, including Toxoplasma gondii, lack CI, raising a fundamental question of how mitochondrial respiration is organized in these parasites. Here, we identify a parasite-specific mitochondrial microdomain that physically and functionally couples ubiquinone (UQ) biosynthesis to succinate dehydrogenase (SDH). Using proximity labeling, membrane-based yeast two-hybrid analyses, reciprocal co-immunoprecipitations, and blue native PAGE, we show that UQ biosynthesis proteins associate with parasite-specific SDH subunits and co-migrate as a high-molecular-weight assembly. Knockdown of TgCoq4, a component of the UQ biosynthesis pathway, reduced SDH activity, and adding exogenous UQ to the assay did not restore it, indicating that UQ availability is not sufficient for efficient succinate oxidation. FCCP stimulates malate- and TMPD/ascorbate-supported respiration to maximal capacity but fails to do so with succinate as the substrate. This result, combined with reactive oxygen species (ROS) data, suggests that succinate causes over-reduction of the UQ pool, limiting further electron flow. We also find that CIII associates with the UQ synthesis machinery, suggesting a larger protein assembly. Together, these findings reveal that UQ biosynthesis and electron transport are physically and functionally integrated in T. gondii, defining a previously unrecognized organization of mitochondrial respiration in an organism lacking CI.

biochemistry↗
bioRxiv2026

Human senataxin uses distinct peripheral elements to unwind R-loops and act on RNA polymerase II

Persistent R-loops and paused or stalled RNA polymerase II (RNAPII) elongation complexes can threaten genome stability. Senataxin (SETX) contributes to R-loop resolution and RNAPII regulation, but their intimate connection during transcription makes the individual contributions of these activities difficult to distinguish in cells. To determine whether these activities rely on the same molecular features, we investigated the structure and biochemical properties of the SETX helicase domain (SETX-HD). Cryo-EM defines the conserved helicase core and RNA-binding channel of SETX-HD, while biochemical analyses identify distinct roles for two peripheral elements: a previously uncharacterised C-terminal RNA-binding domain (CT-RBD) and the prong subdomain. In reconstituted mammalian transcription assays, SETX-HD promotes passage beyond a defined RNAPII pause and elongation-complex dissociation on the same template. The CT-RBD stabilises RNA engagement and is required for efficient R-loop unwinding, whereas its deletion preserves both RNAPII-associated activities. Conversely, replacing the native SETX prong with its fungal Sen1 counterpart preserves R-loop unwinding but impairs both RNAPII outcomes. These reciprocal separation-of-function effects establish that a common SETX-HD RNA-translocation core depends on distinct peripheral elements for R-loop unwinding and action on mammalian RNAPII. This separation provides a direct route to determine how these activities contribute to SETX function in cells.

biochemistry↗