Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

The broad host range bacteriophage Mu employs a novel methylcarbamoyl modification to protect its DNA from diverse host restriction systems. Biosynthesis of the unusual modification is a longstanding mystery. Moreover, isolation of Mom, the phage protein involved in the modification has remained elusive to date. Here, we characterized the co-factor and metal binding properties of Mom and provide a molecular mechanism to explain methylcarbamoylation by Mom. Our computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom, predicting acetyl CoA as its co-factor. We demonstrate that Mom binds to acetyl CoA and identify the active site. Puzzlingly, none of the > 309,000 GNAT members identified so far catalyze Mom-like modification of their respective substrates. Besides, conventional acid-base catalysis deployed by typical acetyltransferases cannot support methylcarbamoylation of adenine seen in Mu phage. In contrast, free radical-chemistry, catalyzed by Fe-S cluster or transition metal ions can explain the seemingly challenging reaction between acetyl CoA and DNA. We discovered that Mom is an iron-binding protein, with the Fe2+/3+ ion colocalized with acetyl CoA in the active site of Mom. Mutants defective for binding Fe2+/3+ or acetyl CoA demonstrated compromised activity, indicating their importance in the DNA modification reaction. Iron-binding in the GNAT active site is unprecedented and represents a small step in the evolution of Mom from the ancestral acetyltransferase fold. Yet, the tiny step allows a giant chemical leap from usual acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture. SummaryStudying the arms race between bacteria and their viruses (bacteriophages or phages) is key to understanding microbial life and its complexity. An unprecedented DNA modification shields phage Mu from bacterial restriction endonucleases that destroy incoming phage DNA. Nothing is known of how the modification is brought about, except that a phage protein Mom is involved. Here, we discover acetyl CoA and iron as key requirements for the modification. We explain how by evolving the ability to bind iron - a transition metal capable of generating highly reactive free radicals, a well-studied scaffold like the acetyltransferase fold can gain novel catalytic prowess in Mom. These findings have broad implications for gene editing technologies and therapeutic application of phages.