SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

Abstract

S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N-and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily. Members of the S-adenosylmethionine (SAM)2 radical superfamily catalyze a wide variety of radical-mediated reactions, including complex chemical transformations and rearrangements; modifications of peptides, DNA, and RNA; dehydrogenations; and sulfur insertions (1). Despite this diversity, there are unifying structural and mechanistic themes. For instance, SAM radical enzymes typically bind a [4Fe-4S] cluster using a conserved CX 3 CXC motif (where is an aromatic residue). This motif provides three cysteine ligands to the iron atoms of the cluster, with the fourth ligand coming from the bidentate coordination of SAM to the unique iron (2, 3). Direct ligation of SAM to the cluster facilitates reductive cleavage of the C-S bond through an inner sphere electron transfer event, forming methionine and a 5Ј-deoxyadenosyl radical (5Ј-dAdo ⅐ ) (Fig. 1A) (4). The abstraction of a hydrogen atom from the substrate by 5Ј-dAdo ⅐ , producing a substrate radical, ends the mechanistic similarity between enzymes of this superfamily; each enzyme utilizes a different mechanism to generate product. Structures of the first seven members of the SAM radical superfamily were used to define a core fold for binding SAM and for the generation of 5Ј-dAdo ⅐ species. This core consists of a partial (␤/␣) 6 triose-phosphate isomerase (TIM) barrel (5). Outside of the core fold, the structure can vary greatly, with Nand C-terminal extensions that are functionalized for binding other cofactors or substrates. The SPASM subfamily is an example of a functionalized C-terminal extension for the binding of two auxiliary clusters.Haft and Basu (6, 7) recognized that enzymes with this C-terminal extension appear to be involved in the modification of ribosomally translated peptides. This subclass is referred to as SPASM after the biochemically characterized members, AlbA, PqqE, anSMEs, and MftC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively. The SPASM subfamily, accession TIGR04085, is composed of 281 sequences. However, recent similarity network analysis by ...

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

“…However, there are known “SPASM” domains that only contain one [4Fe-4S] cluster, which have been dubbed “twitch” domains. 11 Additional studies are required clarify the roles of these auxiliary clusters in AlbA and other SPASM-domain containing rSAM proteins.…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…Electron paramagnetic resonance spectroscopy revealed the presence of two [4Fe–4S] clusters in SkfB. One of the clusters was coordinated by a canonical Cys-containing motif commonly present in radical-SAM enzymes while the second cluster was located within a SPASM (subtilisin A/pyrroloquinoline quinone/anaerobic sulfatase/mycofactocin maturation enzyme) domain (Flühe et al, 2013, Grell et al, 2015, Haft and Basu, 2011). Mutational studies showed one of the [4Fe–4S] clusters to be involved in the generation of the characteristic 5′-dA • radical.…”

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

“…Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8,9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10,11). Recently, a small (ϳ10 kDa) protein, PqqD, has been shown to form a strong, sub-micromolar K D complex with the peptide substrate PqqA.…”