Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family

Abstract

The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis

“…In the only previously known structure of phage-encoded SAM lyase, Svi3-3, the beta strands instead contribute to forming a trimer with active sites at the interface between the subunits. 15 The T3 SAMase binds to MetK using a combination of hydrophobic contacts and hydrogen bonds involving side-chain and backbone atoms (Figure 4D, S5A,B). Residues M1, I2, F3, T4, K5, A8 and N9 from the N-terminal tail of T3 SAMase make contacts with the N-terminal domain and to the linker between the N-and C-terminal domains of MetK, while Q103, T104 and T106 from the β4-β5 loop in T3 SAMase form a network of hydrogen bonds with the N-terminal domain of MetK.…”

“…Prokaryotes developed a broad arsenal of measures to interfere with viral infection. 1 The genomes of most free-living bacteria contain compact defence islands where genes encoding various defence systems are concentrated. 2,3 Likewise, in mobile genetic elements, "anti-defence islands" containing genes responsible for the inhibition of host immunity, for instance, restriction-modification (R-M) or CRISPR-Cas systems, 4 tend to form.…”

“…14 However, recent work demonstrated that T3 SAMase instead cleaves SAM in a lyase reaction (Figure 1A). 15 The T3 SAM lyase is encoded by the 0.3 gene, which is the first gene transcribed upon entry of the T3 genome into the cell. As a consequence, the SAM-cleaving activity is manifested at the earliest stage of phage infection.…”

SAMase of Bacteriophage T3 Inactivates Escherichia coli’s Methionine S -Adenosyltransferase by Forming Heteropolymers

“…S3A ). The topology of the T3 SAMase fold appears to be unique, even compared to a recently solved phage-encoded Svi3-3 SAMase ( 2 ) (PDB entry 6ZMG ) ( Fig. S3B ).…”

“…S -Adenosylmethionine lyase (SAMase) is the first phage-induced protein to appear in T3-infected cells ( 1 ). Although it has long been assumed that SAMase uses water molecules to break SAM into methylthioadenosine (MTA) and homoserine, a recent publication has shown that SAMase is, in fact, not a hydrolase but a lyase degrading SAM into MTA and homoserine lactone ( 2 ). Degradation of the intracellular SAM pools effectively subdues numerous SAM-utilizing reactions of the host cell, including RNA, DNA, protein, and small-molecule methylation, polyamine synthesis, and production of cofactors ( 3 ).…”