Abstract
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.
Highlights
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 50-deoxyadenosyl radical
The high-resolution structure of Arabidopsis thaliana ThiC (AtThiC) co-crystallized with SAH or with 50-dAdo and L-Met showed a consistent arrangement with atoms of SAH closely aligned with the corresponding atoms in 50-dAdo and L-Met (Figs 2f and 3a)
The L-Met amino and carboxylate groups chelate to the additional metal ion and the adenosyl moiety superimposes closely with that of SAH
Summary
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 50-deoxyadenosyl radical. The three cysteine residues ligate three irons of the cluster and SAM is anchored through its amino and carboxylate groups to the fourth iron[11] This canonical mode of SAM binding positions SAM for cleavage of the C50–S bond and generation of the 50-deoxyadenosyl radical and L-Met (Fig. 1a). The radical SAM enzyme PhnJ catalyses cleavage of the C–P bond of phosphonates and has a CX2CX21CX5C motif near the C terminus of the sequence; no structure is available and only recently have the three cysteine residues important for cluster binding been identified[13,14]. We have used AtThiC and CcThiC to determine a series of structures containing the [4Fe-4S] cluster and various combinations of substrates, products and analogues These structures define the fold of the ThiC cluster-binding domain and map out the details of the active site
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