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

Abstract

The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

“…Based on many studies of the chemical and kinetic mechanisms of GNAT enzymes, it is clear this superfamily has evolved to utilize a diversity of chemistries and active site residues for more complex and targeted modifications of acceptor substrates. Identification of additional non-acyl transfer reactions for GNATs, including decarboxylation, methylcarbamoyl transfer, and condensation ( Gu et al, 2007 ; Izoré et al, 2019 ; Karambelkar et al, 2020 ; Skiba et al, 2020 ) highlight the shear multitude of chemistries that enzymes from this superfamily can accomplish. Thus, GNATs appear to have a highly tunable scaffold that has evolved to modify a diverse range of substrates.…”

Modification of DNA by a viral enzyme and charged tRNA

“…S6), the structure shows that Mom forms tight dimers with an interface area of ~850 Å 2 , and a predicted free energy of binding of 4.7 kcal/mol according to the PISA server (32). As anticipated (16,17,31), Mu Mom protomers have the GNAT fold, but with an atypical insertion spanning residues ~175-209 that visually resembles a helixturn-helix (HTH) motif (Fig. 3A-3D) (33)(34)(35).…”

“…1A), reporting an unusual carbamoylmethyl (glycinamide) group at the N6-position of adenine (abbreviated as 6-NcmdA) (13). These foundational studies on Mu also identified a gene, mom (modification of Mu), necessary for the installation of 6-NcmdA during late stages of Mu infection (7)(8)(9)(10)(11)(14)(15)(16)(17). More recently, it was established that mom is the only phage Mu gene required for hypermodification of dA (fig.…”

“…S2-S3, referred to colloquially as ‘momylation’) ( 16–20 ). Protein sequence and structural comparisons showed that Mom belongs to the Gcn5-Related N-Acetyltransferase (GNAT) superfamily ( 16, 18–20 ), though it remained undetermined whether Mom is an active acyltransferase, and if so, which substrate it transfers ( 13, 16, 17 ). Here, we demonstrate that phage Mu enzyme Mom co-opts charged tRNA Gly (Gly-tRNA Gly ) in order to form 6- N cmdA.…”

Evidence of a Set of Core-Function Genes in 16 Bacillus Podoviral Genomes with Considerable Genomic Diversity

“…For example, the last gene in Thornton (gp 43) and SerPounce is functionally annotated as a GNAT family acetyltransferase. These proteins exhibit homology to Escherichia phage Mu methylcarbamoylase mom, which is predicted to modify ~15% of adenine residues in viral DNA to make it resistant to type 1/II restriction enzymes [ 64 ]. A region containing 21 bp of identity was identified on both sides of this gene in the Thornton genome ( Figure 8 c,d), compared to a single copy of this region in Juan.…”