A recent study by Dr. Valakunja Nagaraja and their colleagues at the Indian Institute of Science (IISc, Bangalore), India, discovered a novel strategy by a protein called “Mom” from the bacteriophage Mu that’s crucial for the immune evasion from the restriction enzymes. Dr. Shweta Karambelkar, the lead author of the paper, is currently pursuing her post-doctoral studies at the Bondy-Denomy Lab at UCSF, USA. The novel mechanism of action was found to be modifying the methylcarbomoyl modification of the DNA. In the current study, the authors have studied the metal binding and co-factor requirements and this is the first study to characterize structurally and decipher the active site for binding.
- The methyl carbamoyl adenine modification or ncm6A confers resistance to >20 different restriction enzymes that give the phage to conquer multiple hosts.
- Overexpression of Mom is lethal to E. coli, while the expression in phage Mu during the lytic infection is only for a brief period at the end of its lifecycle.
- The molecular mechanism in which this gene modifies is still unknown due to the absence of structurally or functionally characterized homologs of Mom.
- Cloning of Mom in the T7 expression system and site-directed mutagenesis was performed using inverse PCR, and it’s in vivo activity was measured.
- The in silico prediction of structure and co-factor of Mom was done using Robetta & I-TASSER and CO-FACTOR server respectively to obtain functional insights, including ligand-binding site.
- In the presence of ligands (acetyl CoA, S-adenosyl methionine (SAM), coenzyme A (CoASH), malonyl coenzyme A), the thermostability and binding affinity of Mom were assessed using microscale thermophoresis (MST) along with the salts of various metal ions.
- The cells that expressed Mom remained undigested, while single point mutations in Mom abolished the resistance to HgaI, denoting the specificity of mom activity.
- The Mom comprised of 13% alpha helices and 19% beta-sheets with a molecular mass of 27 KDa existing as a dimer in solution.
- A dose-dependent effect of acetyl CoA binding (Kd ∼ 49 μM) was observed, while no effect for S-adenosyl methionine (SAM), NAD+, glycine, and ATP were observed.
- Mom catalyzes methyl carbamoyl transfer, not acetyl transfer, while it was hypothesized that Mom functioned as a prototypical acetyltransferase.
- Genetic and biochemical approaches were performed for discovering the host proteins that interacted with Mom. The survived colonies were mom+ but defective of DNA modification.
- In a protein-protein interaction, assay revealed ferritin and subunits of the pyruvate dehydrogenase (PDH) complex to the partners.
- The colorimetric assay revealed Fe2+/3+ bound to Mom but not to other cations such as Mn2+, Cu2+, Mg2+, Co2+, Zn2+, and Ca2+.
- Further, six-point mutants (H48A, Y49A, D139A, Y149A, S114A, and R101A) were tested for defects in Fe2+/3+ and/or acetyl CoA-binding, and they fell into four categories based on defects in (i) acetyl CoA-binding only, (ii) both iron- and acetyl CoA-binding, (iii) iron-binding only and (iv) neither iron-binding nor acetyl CoA-binding.
- The mechanism of the unusual DNA modification in phage Mu has been deciphered, and they discovered the acetyl CoA and Fe2+/3+ to be the co-factors with novel catalytic functions.
- These findings pave the way for the potential of the therapeutic application of phages.