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

Abstract

The first SAM degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as an S adenosyl-L-methionine (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 NMR 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, in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

New views on PII signaling: from nitrogen sensing to global metabolic control

“…Quite strikingly, the first PII-like protein with enzymatic activity has been identified in a bacteriophage and shown to catalyze lysis of S-adenosylmethionine (SAM). The catalytic site of this PII-like SAMase is located in the lateral clefts between the subunits, resembling the nucleotide-binding pockets of canonical PII [83]. The ability of various PII-like proteins to bind/hydrolyze adenine-based nucleotides concurs with the hypothesis that the primordial function of ancestral proteins of the PII superfamily was likely the interaction with adenine nucleotides, which was later extended in (most) canonical PII proteins by additional interaction of 2-OG with Mg 2+ -ATP-ligated PII [6,8].…”

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

“…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 inactivatesE. coli’smethionine S-adenosyltransferase by forming hetero-polymers

“…Two T3 SAMase monomers interact along ~1,000Å 2 isologous interface to form a C2-dimer. The MAT-SAMase interface is smaller and occupies ~600-700Å 2 (see Methods and Table S3). The "cracked egg" structure of SAMase dimer pushes one of the two joined MAT tetramers away from the plane (Fig.…”

“…S-adenosylmethionine lyase (SAMase) is the first phage-induced protein to appear in the T3infected 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).…”