Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

Abstract

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S:-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

Figure 1

PROBE alignment of Radical SAM superfamily members. The PROBE iterative profile tool is used to represent the distant sequence similarity between Radical SAM proteins in the form of alignment blocks. Only the strongest regions of the strongest blocks are displayed. Additional motifs emerge in sub-categories of proteins. The sequences included by PROBE in the alignment model represent every group in the BLAST distance dendrogram at the level of 29 clusters and 48 groups at the level of 50. The bars above the sequences show the information content of the PROBE model at each residue position, ranging from 0 to 5 bits. Above the sequences are shown individual residues with the highest information value in each position. Residues are marked in bold for positions with information content of 2 bits or greater. Also marked in bold are aromatic residues (Y, F and W) adjacent to the third cysteine in the first motif, as well as glycine and proline residues (G and P) in the second motif. Each protein is labeled on the left with a gi number, identifying the sequence in the GenBank database, and on the right with a protein name or pathway when known. The coordinates at the left of the alignment blocks suggest the size of independent N-terminal domains and, at the right, the size of C-terminal domains when compared against the sequence length.

Figure 2

Dendrogram visualization of Radical SAM core domains. The sequence similarity relationships in the superfamily are visualized using the mBE measure of cluster cohesion to filter a dendrogram produced by hierarchical clustering of a distance matrix. On the left is the line drawing of the original dendrogram, which conveys a representation of between cluster distances of the basic groups. On the right, with increasing cluster number, colored bars are superimposed over the most complex region of the original line drawing using an mBE threshold value applied to the groups. A colored bar appears when a group of sequences appears at a node with an mBE value of <1.0. Further divisions within groups are represented by the color scheme, with cool colors representing looser groups and warm colors tighter ones. Many nodes of the dendrogram are labeled with the number of clusters found at that level. The right edge of the dendrogram is at 200 clusters. Groups discussed in the domain and operon analyses are highlighted with a white background. The ExsD and the Pyrococcus furiosus protein groups from the operon discussion are labeled with asterisks. The color scale for mBE intervals is as follows: blue (1 > E > 1e-6); green (1e-6 > E > 1e-12); yellow (1e-12 > E > 1e-24); orange (1e-24 > E > 1e-48); red (1e-48 > E). Many small branches of less interest have been ‘closed’ (represented by grey bars) to place distracting details in the background.

Figure 3

Multiple alignment of the Radical SAM N-terminus and cobalamin-binding domains. A multiple alignment between cobalamin-binding proteins and a subset of Radical SAM N-terminal domains shows that Motif 2 but not Motif 1 is conserved between the two groups. Motif 1 is the ‘base-off’ consensus that supplies a histidine residue to displace the dimethylbenzimidazole moiety from cobalamin. The conserved Motif 2 residues are known to play a role in binding cobalamin in these corrinoid proteins. The Radical SAM proteins are a bacteriochlorophyll biosynthesis protein BchE (gi|114858), a putative methyltransferase (gi|6686119), a fortimicin methyltransferase (gi|1125024), a fosfomycin methyltransferase (gi|2144248), a bialaphos P-methylase (gi|529098), the mitomycin C biosynthesis protein MmcD (gi|4895120) and the oxetanocin biosynthesis protein OxsB (gi|7474372). The corrinoid-binding proteins are CMT (corrinoid methyltransferase, gi|7483270), DMT (dimethylamine corrinoid protein, gi|4262424), MS (methionine synthase, gi|400244), MGM (2-methyleneglutarate mutase, gi|543481), MCM (methylmalonyl-CoA mutase, gi|1942488) and GM (glutamate mutase, gi|7245512).