MenT nucleotidyltransferase toxins extend tRNA acceptor stems and can be inhibited by asymmetrical antitoxin binding

Abstract

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.

Nucleotidyltransferase toxin MenT targets and extends the aminoacyl acceptor ends of serine tRNAs in vivo to control Mycobacterium tuberculosis growth

“…M. tuberculosis encodes four MenAT TA systems, comprised of a nucleotidyltransferase (NTase) toxin and a cognate antitoxin belonging to one of three different families (Dy et al, 2014;Xu et al, 2023;Cai et al, 2020). Although, in vivo, menAT2 was recently shown to be required for M. tuberculosis pathogenesis in guinea pigs (Gosain et al, 2022), only menAT1 and menAT3 were shown to act as bona fide TA systems in their native host M. tuberculosis (Xu et al, 2023;Cai et al, 2020).…”

“…tuberculosis encodes four MenAT TA systems, comprised of a nucleotidyltransferase (NTase) toxin and a cognate antitoxin belonging to one of three different families (Dy et al, 2014;Xu et al, 2023;Cai et al, 2020). Although, in vivo, menAT2 was recently shown to be required for M. tuberculosis pathogenesis in guinea pigs (Gosain et al, 2022), only menAT1 and menAT3 were shown to act as bona fide TA systems in their native host M. tuberculosis (Xu et al, 2023;Cai et al, 2020). The MenA3 antitoxin inhibits MenT3 through phosphorylation of a serine residue in the catalytic site (Yu et al, 2020), whilst MenA1 forms an asymmetric heterotrimeric complex with two MenT1 protomers, suggesting a different mode of inhibition (Xu et al, 2023).…”

“…Although, in vivo, menAT2 was recently shown to be required for M. tuberculosis pathogenesis in guinea pigs (Gosain et al, 2022), only menAT1 and menAT3 were shown to act as bona fide TA systems in their native host M. tuberculosis (Xu et al, 2023;Cai et al, 2020). The MenA3 antitoxin inhibits MenT3 through phosphorylation of a serine residue in the catalytic site (Yu et al, 2020), whilst MenA1 forms an asymmetric heterotrimeric complex with two MenT1 protomers, suggesting a different mode of inhibition (Xu et al, 2023). In vitro, the MenT1, MenT3 and MenT4 toxins were shown to inhibit translation by acting as tRNA NTases (Xu et al, 2023;Cai et al, 2020).…”

“…The MenA3 antitoxin inhibits MenT3 through phosphorylation of a serine residue in the catalytic site (Yu et al, 2020), whilst MenA1 forms an asymmetric heterotrimeric complex with two MenT1 protomers, suggesting a different mode of inhibition (Xu et al, 2023). In vitro, the MenT1, MenT3 and MenT4 toxins were shown to inhibit translation by acting as tRNA NTases (Xu et al, 2023;Cai et al, 2020). Yet, biochemical characterization revealed significant differences in MenT toxin specificity for tRNA targets and nucleotide substrates.…”

“…Yet, biochemical characterization revealed significant differences in MenT toxin specificity for tRNA targets and nucleotide substrates. Though both MenT1 and MenT3 inhibit aminoacylation by transferring pyrimidines (preferentially cytidines) to the 3′ CCA acceptor stems of tRNAs, MenT3 displays a strong preference for serine tRNAs and MenT1 showed no apparent preference (Xu et al, 2023;Cai et al, 2020). MenT4 also modifies the 3′ CCA motif of tRNA acceptor stems but with a preference for GTP as substrate (Xu et al, 2023).…”