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Nucleic Acids Res. 2001 March 1; 29(5): 1097–1106. | PMCID: PMC29726 |
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 Heidi J. Sofia,1,2a Guang Chen,3 Beth G. Hetzler,4 Jorge F. Reyes-Spindola,5 and Nancy E. Miller4 1Applied Mathematics, Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory, Richland, WA 99352, USA, 2Biology Department, Whitman College, Walla Walla, WA 99362, USA and 3Statistics Resources, 4Synthesis, Analysis and Visualization of Information (SAVI) and 5Information Sciences and Engineering, Pacific Northwest National Laboratory, Richland, WA 99352, USA Received November 15, 2000; Revised January 9, 2001; Accepted January 9, 2001. 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. Sophisticated iterative profile methods have dramatically extended
the power of sequence homology searches ( 1– 3). These tools are useful for creating
a larger context for database search results. Whereas a strong match
in a BLAST search can be used to infer similar function, the weaker
similarity detectable by an iterative profile method illuminates
a more distant relationship and is evidence of a conserved fold
in the protein structure ( 2).
An anonymous sequence without significant pairwise similarity can
often be linked in this way with proteins that have been characterized
experimentally ( 4). Iterative profile searches are easy to perform but can be difficult
to interpret because the data sets returned are large. A query is
linked to numerous sequences, each with multiple links to other
data sources, creating a large information landscape that can be
hard to navigate. As a result, when performing iterative profile
searches on the most interesting and novel sequences, a scientist
is likely to be overwhelmed with data presented simply as long linear
lists, a sharply limited view of information that is inherently
multi-dimensional. We have applied powerful bioinformatics and information visualization
techniques to overcome these obstacles in the analysis of an important
new protein superfamily that we discovered using iterative profile
searching. We call this new superfamily Radical S-adenosylmethionine
(SAM) after the defining characteristics of its best-studied members.
Radical SAM is an ancient and diverged group with 645 unique sequences
from 126 species found to date from all three domains of life. At
least half the proteins are of unknown activity. We use exploratory
statistical methods to analyze the sequence similarity relationships
and integrate these results with other data types (motif, domain,
operon structure, biochemical pathway and the biomedical literature)
for discovery efforts on previously uncharacterized sequences. Our
results are part of a larger effort to scale up biological knowledge
production using four accelerating factors: (i) information visualization;
(ii) large computational resources; (iii) new mathematical strategies;
(iv) collaborative problem solving environment technology. Detection of the superfamily The reader can directly observe evidence for distant sequence similarity
in the Radical SAM superfamily using the Web version of PSI-BLAST
( http://www.ncbi.nlm.nih.gov/BLAST/) at
the National Center for Biotechnology Information (NCBI, Bethesda,
MD). For example, enter any gi identifier from Figure (such as 128228 for the Azotobacter
vinelandii NifB protein) and iterate to convergence with the
default threshold. In this work PSI-BLAST ( 2)
searches were performed locally using command line searches against
the non-redundant protein database downloaded from NCBI onto a Sun
Ultra 60 workstation. Search results were analyzed and
tested for the closure property with standard Unix tools. The 54
sequences directly tested include 30 proteins associated with at
least some biochemical information as well as others chosen to represent the
most distant members. False negatives for any individual search
were measured against the set of unique and complete sequences already
accepted as belonging to the superfamily from multiple other searches.
False positives were measured against a list that also included
redundant sequences and fragments. False positives ranged from 0
to 12%, with a median value of 0.2%. False negatives
ranged from 0.7 to 16%, with a median value of 3%.
There were seven sequences that required either an increased or
decreased threshold [from the default Expect ( E)
value of 0.001] for convergence to occur. (Searches that
fortuitously include significant numbers of unrelated sequences
in a profile do not converge but rather ‘explode’ and
can pull in the entire database.) With the 15 N-terminal/motif deletion
sequences the median rate of false positives was unchanged, but
false negatives increased to 5%. PROBE ( 3)
is a powerful iterative profile search tool that was used in this case
as a convenient method for extracting alignment blocks from the
defined set of highly diverged Radical SAM proteins. These blocks
were edited to show the strongest conserved regions. BLAST E scores are reported in the computer
style of standard scientific notation (e.g. 3e-20 represents
3 × 10–20). Analysis and visualization of superfamily data Standard Unix tools, S-PLUS (MathSoft, Cambridge, MA), the OmniViz
Pro software package (OmniViz, Richland, WA) and custom Perl programs
were used for superfamily analysis. At the time the analysis was
initiated there were 533 unique and complete Radical SAM proteins
in the database. The conserved core domains (estimated at ~200
residues and starting at the conserved cysteine motif) were extracted
from the Radical SAM sequences using an S-PLUS script. A Perl program
was used to perform a complete BLAST comparison of the core domains
to produce a matrix of BLAST E values with a high
score cut-off of 1000 and then to transform the matrix (lowest score
of 0 set to 1e-200, all missing scores to 10 000
and take logn). The transformed matrix was then imported
into OmniViz Pro for hierarchical clustering (complete linkage with
Euclidean distance). Data files produced by OmniViz Pro were imported
into S-PLUS to produce a preliminary dendrogram representation.
Cluster membership in the dendrogram was analyzed by making a Galaxy visualization
in OmniViz Pro at each level of interest for the purpose of capturing
the cluster membership list. These lists were examined individually
and were also combined into a spreadsheet for a convenient view
of the data. Inclusion of the location of the cysteine motif and
the size of the proteins in the spreadsheet allowed patterns to
be detected in the size of N- and C-terminal domains. The dendrogram
visualization was created with an S-PLUS program that generated
the colored blocks from the hierarchical clustering results and
Adobe Illustrator (San Jose, CA). The NCBI Web Entrez interface was used for access to MEDLINE
and large sets of abstracts were downloaded using Network Entrez
for analysis with the SPIRE technology ( http://multimedia.pnl.gov:2080/infoviz/technologies.html),
which provides an interactive topic map based on word frequency analysis. Discovery of a novel superfamily A small collection of proteins with diverse functions have been noted
to share an unusual Fe-S cluster associated with generation of a
free radical by reductive cleavage of SAM. This
group consists of lysine 2,3-amino mutase (LAM), biotin synthase (BioB),
lipoic acid synthase (LipA) and the activating proteins for pyruvate
formate-lyase (PflA) and anaerobic ribonucleotide reductase (NrdG).
These ‘deoxyadenosyl radical’ enzymes have been
the focus of detailed experimental work, including UV-Vis, EPR,
Mössbauer, resonance Raman, variable temperature magnetic
circular dichroism and mutagenesis experiments ( 5– 12). SAM has been described as equivalent
to a ‘poor man’s coenzyme B 12’ in
the reaction catalyzed by LAM ( 13).
Very recently, K edge X-ray absorption spectroscopy experiments
have provided important mechanistic evidence for the direct role
of the unusual Fe-S cluster in LAM in the reductive cleavage of
SAM ( 14– 16). Despite the attention they have received, the deoxyadenosyl radical
proteins have not been previously recognized as homologous sequences,
although a characteristic cysteine motif has been noted ( 7). We applied sensitive bioinformatics methods
that detected distant sequence similarity between these five protein
groups. This observation is evidence for a shared ancestor and supports
the prediction of a common fold for the core domain. Our results
also link these enzymes to a larger collection of known and unknown
functions, a list that includes proteins found at unresolved steps
in familiar biosynthetic pathways, such as thiamin, heme, heme d1,
bacteriochlorophyll, molybdopterin, nitrogenase cofactor, pyrroloquinoline quinone,
desosamine and others in secondary metabolism. We detected distant sequence conservation between the Radical
SAM proteins with PSI-BLAST iterative profile searching ( 2). We observed that these proteins form
a closed set with the following property. Each sequence detects
the same hit list within a small margin of error after iteration
to convergence with a conservative threshold (for details see Materials
and Methods). Proteins classified as belonging to the superfamily
were either directly tested for this closure property (54 sequences)
or shown to be strongly similar to one that was. All of the 645
unique and complete sequences collected in this manner were observed
to contain an unusual conserved cysteine motif, most often near
the N-terminus or in some longer sequences in the middle. These
include 592 proteins with an exact match to the consensus CxxxCxxC
and 53 variants with a small increased distance between the first
two cysteine residues. We also tested 15 diverse Radical SAM proteins after removal
of the N-terminus including the cysteine motif. This deletion had
the effect of reducing the sensitivity of the searches, but not
the specificity. Interestingly, the oxygen-sensing regulatory protein
FNR has been described as containing an Fe-S cluster similar to
those found in the deoxyadenosyl radical proteins both in the cysteine
motif and in a reversible transition from [2Fe-2S] 2+ to [4Fe-4S] 2+ controlled by
the presence of oxygen ( 16, 17). However, FNR proteins were never
detected in any of the Radical SAM searches. Therefore, the presence
of the cysteine motif is not necessary or sufficient for inclusion
in the superfamily by PSI-BLAST detection of distant sequence similarity. We used the PROBE ( 3) software
against the diverged set of Radical SAM sequences to extract alignment
blocks and show the strongest sections of these in Figure . The cysteine motif in the first block has
the highest information density (in units of bits). A conserved
aromatic residue (Y, F or W) adjacent to the third cysteine may
function to lower the midpoint potential of the cluster by limiting
solvent exposure ( 16). The second block
contains a glycine-rich sequence resembling the SAM-binding site
in methyltransferases ( 18) and
could play a role in binding this molecule for reductive cleavage. Protein sequences evolve more quickly than the corresponding three-dimensional
structures and, as our results illustrate, proteins with a common
fold may only show faint sequence conservation that approaches the
limit of detection. However, these patterns can be extracted with
sensitive bioinformatics approaches and the information they contain
has quantitative value, as exemplified by recent successes in ‘threading’ protein fold
prediction programs that include PSI-BLAST results as a term in
the calculations ( 19). Organizing and visualizing superfamily relationships The Radical SAM classification places 645 proteins into a single
conceptual box but does not illuminate any details of how the members
are organized. Although a phylogenetic tree is a useful way to analyze
a sequence family, it is difficult to create a multiple alignment
for this purpose with highly diverged proteins ( 20).
We applied clustering, a well-known approach in exploratory statistics
for extracting groups, to characterize the sequence similarity relationships
between the Radical SAM core domains and generate a dendrogram ( 21). In this approach we used a tree representation
not to represent phylogeny but rather to display sequence similarity
relationships between superfamily homologs. We first generated a
feature matrix based on complete BLAST comparisons between the conserved
core domain in each Radical SAM member. We then used hierarchical
clustering (complete linkage with Euclidean distance) on the BLAST E score feature matrix to organize the sequences
and produce the dendrogram. This algorithm results in a hierarchical
tree with the property that variance within each cluster is minimized.
We found this preliminary dendrogram to be useful in many ways,
for example in identifying misnamed sequences, classifying unknown
sequences and in supporting the definition of unique features that
characterize sub-groups. However, navigating the raw dendrogram
with its associated lists of groups was a difficult and rate-limiting
step in the analysis. We therefore created a visual prototype for
an automated and interactive solution. Visual display of information is considered a ‘broad
bandwidth’ pathway to the human brain. Powerful visual
problem solving approaches have been applied to the analysis of complex
hierarchical data ( 22– 28). We used aspects of this research
to create a new visual representation for our data. We applied a
measure of cluster cohesion that we have named the maximum BLAST E (mBE)
score to the clusters in the dendrogram as the basis for color coding
groups of closely related proteins that may share a common function
(Fig. ). This mBE cluster cohesion metric
is defined as the maximum of the BLAST E values
in the subset of the original feature matrix defined by the proteins
in the group under consideration. Therefore, our measure of the ‘tightness’ of
any cluster is directly based on the largest of all the BLAST comparisons
for the proteins in a group. Strong relationships in the dendrogram are
depicted with colored blocks that appear when the mBE value is <1.
Further divisions within groups are represented by the color scheme,
with cool colors representing looser groups and warm colors tighter
ones. This visual encoding essentially creates a level of abstraction
on distracting details and facilitates interpretation of the results. For example, at the level of eight clusters in the dendrogram a
group of 40 HemN-related sequences appear with an mBE value of 0.028
(Fig. and Table 1).
This group, shown in blue (a cool color reflecting lower cohesion)
divides again at 37 clusters, producing 19 HemN sequences
(mBE 3e-24, yellow) and 21 HemN-like sequences (mBE 1e-8,
green). Examining the sequences, we observed that the HemN proteins all
contain an extra cysteine at the end of the conserved motif (CxxxCxxCx C) but the HemN-like proteins, such as in Bacillus
subtilus ( 29), and the PhuW,
HutW and ShuW virulence proteins ( 30)
do not. Thus, our visual algorithm presents these two related but
distinct groups of proteins in an intuitive fashion and facilitates
integration of this motif information into the analysis. Interactive visualization strategies are beginning to be part
of the analyst’s basic toolkit in working with large-scale
information. We rely on the SPIRE technology ( 31)
to explore large sets of biomedical records through interactive
topographical maps based on word frequency statistics ( http://multimedia.pnl.gov:2080/infoviz/technologies.html).
In a similar way, we envision an automated and interactive version
of our dendrogram visualization as a data mining tool that supports
biological problem solving by creating a map of superfamily sequences
and providing a framework for the integration of diverse data types in
the analysis of unknown proteins. Radical SAM superfamily proteins We explored the organized view of the Radical SAM proteins to
find 30 distinct groups associated with at least some biochemical
data (Table 1). Interestingly, the most
distantly related clusters (diverging first in the hierarchical
tree) seem to share an involvement with sulfur transfer; these include
the NifB, MiaB, BioB and LipA proteins ( 7– 9, 32– 35). A mechanism for sulfur transfer from
the Fe-S cluster in BioB has been proposed ( 32).
Like biotin synthase, the NifB proteins act as reagents and not
catalysts in existing in vitro assays ( 7, 33).
A group of 53 sequences, including MiaB (appearing at the level of
three clusters with an mBE value of 0.001), also contains a novel
human Cdk5 activator-binding protein that binds the neuronal Cdc2-like
kinase (Nclk) involved in regulation of neuronal differentiation
and neuro-cytoskeletal dynamics ( 36). Other Radical SAM proteins, such as ThiH of thiamin and MoaA
of molybdopterin biosynthesis, are found in pathways with sulfur
transfer, but most likely do not act in this role directly. Interestingly,
however, the MoaA proteins contain the residues GG at the C-terminus,
a motif that is adenylated for sulfur transfer in the MoaD proteins,
as in ubiquitin ( 37). In thiazole
biosynthesis, sulfur is mobilized from cysteine in a manner similar
to the molybdopterin pathway, with adenylation/thiocarboxylate
formation at a C-terminal GG motif in ThiS ( 32). Radical SAM proteins often provide an anaerobic or oxygen-independent
mechanism that is found as an aerobic reaction in other proteins,
for example HemN ( 38), BchE ( 39) and, possibly, ThiH ( 32).
The HemN protein catalyzes an oxygen-independent oxidation in anaerobic
heme biosynthesis and has been shown to require NADH and either
SAM or ATP and methionine for in vitro activity
( 40), which Thauer is reported to
have explained with the hypothesis of deoxyadenosyl radical chemistry
( 38). In heme d1 biosynthesis
the anaerobic production of oxo groups at positions C3 and C8 is
of special interest as a possible role for the NirJ protein ( 41). The oxidation of serine or cysteine
to formylglycine is catalyzed by sulfatase activation proteins similar
to AslB; the activation of arylsulfatase has been observed under
both aerobic and anaerobic growth conditions ( 42, 43). Radical SAM proteins are associated with several ring-forming reactions.
ThiH functions in thiazole ring formation from tyrosine, cysteine
and 1-deoxy- d-xylulose-5-phosphate ( 32),
PqqE in cyclization of the tyrosine amino acid backbone with glutamate addition
to form the cofactor PQQ ( 44)
and BchE in isocyclic ring formation in bacteriochlorophyll ( 39). The MitD protein in the mitomycin
C gene cluster may catalyze mitosane ring formation from the condensation
of 3-amino-5-hydroxybenzoic acid, d-glucosamine
and carbamoyl phosphate ( 45). Three eukaryotic interferon-inducible members are found, including
a rat gene (best5) expressed during osteoblast differentiation and
bone formation and a candidate drug target for osteoporosis ( 46), a human gene (cig5) induced during cytomegalovirus
infection ( 47) and a trout gene
(vig1) induced during rhabdovirus infection ( 48). Many examples from secondary metabolism pathways, such as antibiotic
and herbicide biosynthesis, are found, including spectinomycin ( 49), subtilosin ( 50),
nikkomycin ( 51), mitomycin C
( 45), oxetanocin ( 52),
fortimicin, fosfomycin and bialaphos biosynthesis ( 53, 54) and the desosamine moiety of erythromycin
( 55), oleandomycin ( 56), methymycin, neomethymycin, narbomycin
and pikromycin ( 57). Biodegradation
is represented by BssD in toluene catabolism ( 58)
and DNA repair by spore photoproduct lyase ( 59). Two functional predictions for unknown proteins Problem solving in the genomics era increasingly depends upon
traversing complex data landscapes with computational and visualization
approaches. We present two examples of functional prediction for
unknown proteins in the Radical SAM superfamily based on a multi-dimensional
approach to data mining. These analyses were performed in a semi-manual
fashion as a preliminary effort in the large-scale automation of
the approach. Although a dendrogram for the Radical SAM core domains is a
useful tool for classification and annotation, it essentially provides
only a one-dimensional analysis of the superfamily proteins based
on the single data type of sequence similarity. Features such as
motif, domain, operon, biosynthetic pathway, chemical structure
and properties described in the biochemical literature can all provide
important functional clues to the biologist when viewed in an organized
context. We use the similarity dendrogram as a framework for the
integration of multiple data types for the purpose of gaining leverage
in the functional prediction of unknown proteins. Example 1. Many Radical SAM sequences have independent N-
or C-terminal domains (Fig. ). We correlated
the sizes of these independent domains with cluster membership in
the dendrogram. For example, at the level of 41 clusters in the dendrogram
(Fig. ) a group of 26 proteins appears
that shows poor cohesion even after multiple divisions, a feature
that often suggests divergent functions. However, 25 of these proteins possess
a long N-terminal extension of ~200
residues. PSI-BLAST searches show that these N-terminal sequences
have distant sequence similarity (Fig. ).
The proteins in this group, linked by both cluster membership and
a shared N-terminal domain, include the fortimicin (FMT) and fosfomycin
(PAMT) methyltransferases ( 53, 54), OxsB ( 52)
(oxetanocin) and MmcD ( 45) (mitomycin
C). Also sharing the N-terminal domain but located in different
clusters are the BchE proteins ( 38, 39) and the bialaphos P-methylase (PMT)
( 53, 54). With further PSI-BLAST iterations of the N-terminal domain (with
BchE for example) proteins outside the Radical SAM superfamily are
detected, which share the property of binding cobalamin ( 60; Fig. ). This
result is interesting in the light of experimental evidence that
the fortimicin, fosfomycin and bialaphos methyltransferases ( 53, 54)
and, recently, BchE ( 61) utilize
a cobalamin cofactor. Methylation reactions commonly occur through
electrophilic attack of a methyl cation. However, the fortimicin,
fosfomycin and bialaphos biosynthesis proteins each transfer a methyl
group to an electrophilic site ( 54).
Taken together, these data (dendrogram, domain and biochemical)
reinforce each other and suggest that the candidate methyltransferase
proteins found at cluster level 41 in our analysis are likely to
share an unusual chemical mechanism even as they have diverged in
sequence as a result of acting on distinct substrates and pathways. Example 2. Operon data can be powerfully integrated
with clustering results and biochemical data in a similar way. Little is
known about the Radical SAM member ExsD ( 62)
except that proteins in this operon impact on succinoglycan biosynthesis.
We used the neighboring ExsC protein to search an operon database
(made by extracting protein links from Radical SAM nucleotide records)
as a means of finding other superfamily members with this linkage.
ExsC homologs are found adjacent to nine Radical SAM proteins, located
in two clusters (Fig. ), one containing
ExsD and another with a Pyrococcus furiosus protein
( 63). Therefore, it appears likely that
the location of ExsD next to ExsC is not fortuitous. These results
suggest an interesting hypothesis. ExsC is strongly related to 6-pyruvoyl
tetrahydrobiopterin synthase, the second step in tetrahydrobiopterin
biosynthesis ( 64). The first
step in tetrahydrobiopterin biosynthesis is the production of a
pterin ring by GTP cyclooxidase I. Interestingly, the MoaA proteins provide
a unique mechanism for production of a pterin ring from GTP in molybdopterin
biosynthesis ( 65). Therefore,
by analogy, the ExsD Radical SAM protein and its neighbor in the operon
ExsC could be the first two steps of an unusual pterin synthesis
pathway. With over 600 unique sequences, 30 known functions and many additional
unknowns, the existing biochemical and genetic data on the Radical
SAM proteins easily represent over 1 000 000 person-hours
of experimental work in the laboratory. With identification of the
superfamily this knowledge base becomes a resource supporting the
laboratory efforts of a newly defined community of experimental
scientists. All the Radical SAM proteins can now be evaluated for
radical chemistry as well as other properties. The usefulness of
the classification is illustrated by experiments performed by Nicholson
and co-workers based on the observation that spore photoproduct
lyase contains the characteristic cysteine motif of the deoxyadenosyl
radical proteins ( 59). They modified
an assay for anaerobic ribonucleotide reductase and successfully measured
spore photoproduct lyase activity in vitro for
the first time. Radical SAM represents a mechanistic solution for the catalysis
of difficult chemical reactions. Robert H. Abeles, who uncovered
many unusual enzymatic reactions, is reported to have said ‘if
you can formulate on paper a mechanism in two-electron steps, then
there is no radical involved’, and this comment is still
a practical one ( 66). However,
the many two-electron mechanisms proposed for proteins in this new
superfamily can now be seen as too conservative and can be reasonably made
more radical. We thank Gus J.Calapristi and Jim J.Thomas for helpful discussions
and Wanda F.Mar and Leigh K.Williams for technical assistance. This
work was performed at the Pacific Northwest National Laboratory
(PNNL), which is operated by Battelle for the US Department of Energy.
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