Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix

Abstract

Clostridioides difficile infections are an urgent medical problem. The newly discovered C.difficileadenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-l-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

Structural basis for MTA1c-mediated DNA N6-adenine methylation

“…The MTA1c complex also shows significant structural and mechanical differences from the bacterial DNA adenine methyltransferases, such as M.TaqI, M.EcoP15I, Dam, CcrM, and CamA, whose structure and catalytic mechanism have been well studied 19 – 23 . First, these bacterial enzymes are active as monomers or homodimers, while the MTA1c complex functions as heterotetramer.…”

“…SAM-dependent methyltransferase activity assay was displayed by using the MTase-Glo TM assay as described previously 19 , 43 , 44 . Briefly, an assay cocktail was prepared with 20 mM Tris, pH 7.5, 200 mM NaCl, 6 mM EDTA, 5 μM enzyme and 2.7 μg of 954-bp dsDNA substrate (or 50 μM 59 bp oligo dsDNA:5′-AACTTCTGTCATTACATTAAGCTTTAAAAAATTCAATTCTTTTATTTATTAGAATTATG-3′) was added in a 0.2 ml PCR tube, then 160 μM SAM was added to initiate the reaction with a final assay volume of 50 μl for 4 h at 37 °C.…”

“…Writers are the DNA methyltransferases (MTases) that transfer a methyl group from S-adenosyl methionine (SAM) to the N6 position of an adenine residue to form 6 mA. In prokaryotes, multiple DNA adenine methyltransferases, such as CcrM, M.EcoP15I, M.TaqI, CamA and Dam, have been well studied 19 – 23 . M.TaqI, CamA and Dam are active as monomers, while CcrM and M.EcoP15I function as dimers, similar to eukaryote DNA cytosine methyltransferases Dnmt3a/3b/3L and DRM2, and RNA adenine methyltransferase METTL3-METTL14 24 – 27 .…”

Bacterial Epigenomics: Coming of Age

“…One interesting possibility for MTase inhibition would be to use analogs of the methyl donor S -adenosyl- l -methionine (SAM) or bisubstrate inhibitors that simultaneously target SAM- and substrate-binding sites ( 17 ). Such a scenario was recently proposed for the core MTase CamA of C. difficile ( 18 ). Another example is that of the well-characterized Escherichia coli Dam enzyme, whose inhibition reportedly weakens bacterial pathogenicity in vivo ( 19 , 20 ).…”

Repurposing epigenetic inhibitors to target the Clostridioides difficile-specific DNA adenine methyltransferase and sporulation regulator CamA

“…The catalytic domain of CamA [ 37 ] is consistent with the Rossman-fold of Class I MTases [ 38 ], which share a conserved SAM binding site ( Figure 1 ). This structure is shared by (among many other MTases) human DOT1L (a histone H3 lysine-79 MTase [ 39 ]) and PRMTs (protein arginine MTases [ 40–43 ]).…”