Download Structure and function of radical SAM enzymes

Structure and function of radical SAM enzymes
Gunhild Layer, Dirk W Heinz, Dieter Jahn and Wolf-Dieter Schubert
‘Radical SAM’ enzymes juxtapose a [4Fe-4S] cluster and
S-adenosyl-L-methionine (SAM) to generate catalytic
50 -deoxyadenosyl radicals. The crystal structures of
oxygen-independent coproporphyrinogen III oxidase HemN
and biotin synthase reveal the positioning of both cofactors
with respect to each other and relative to the surrounding
protein environment. Each is found in an unprecedented
coordination environment including the direct ligation of
the [4Fe-4S] cluster by the amino nitrogen and one
carboxylate oxygen of the methionine moiety of SAM, as
observed for other members of the Radical SAM family by
ENDOR. The availability of two protein structures supported by
biochemical and biophysical data underscores common
features, anticipating the structural elements of other family
members. Remaining differences emphasize the plasticity
of the protein scaffold in functionally accommodating 600
family members.
Addresses
Divison of Structural Biology, German Research Center for
Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig,
Germany
e-mail: [email protected]
Current Opinion in Chemical Biology 2004, 8:468–476
This review comes from a themed section on
Mechanisms
Edited by Hung-Wen Liu and Jànos Rétey
Available online 20th August 2004
1367-5931/$ – see front matter
# 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2004.08.001
Abbreviations
aRNR-AE
anaerobic ribonucleotide reductase activating enzyme
BioB
biotin synthase
BssD-AE
benzylsuccinate synthase activating enzyme
CPO
coproporphyrinogen III oxidase
DhaB2
B12-independent glycerol dehydratase activating enzyme
ENDOR
electron nuclear double resonance
EPR
electron paramagnetic resonance
HemF
oxygen-dependent coproporphyrinogen III oxidase
HemN
oxygen-independent coproporphyrinogen III oxidase
LAM
lysine 2,3-aminomutase
LipA
lipoate synthase
PFL-AE
pyruvate formate-lyase activating enzyme
SAM
S-adenosyl-L-methionine
SPP lyase spore photoproduct lyase
Introduction
Radical enzymatic reactions, although quite rare compared with their non-radical brethren, are increasingly
Current Opinion in Chemical Biology 2004, 8:468–476
recognized to be integral to the mainstream biosynthetic
arsenal of living organisms — frequently reserved for
some of the most difficult chemical reactions [1]. Most
radical enzymes generate their own organic radicals.
Many, however, rely on activating enzymes (activases)
to provide the organic radical, storing it as remarkably
stable glycyl, cysteinyl, tryptophanyl or tyrosinyl radicals
for repeated catalytic turnovers. Mechanisms of catalytic
free radical generation include photoactivation of cofactors, interaction of molecular oxygen with non-heme diiron, with heme or Cu-tyrosine centers and the homolytic
cleavage of the C–Co bond of the cofactor adenosylcobalamin (B12). Yet a further mechanism, chemically
related to that of adenosylcobalamin in that a 50 -deoxyadenosyl radical is generated, requires the juxtaposition
of a [4Fe-4S] cluster and S-adenosyl-L-methionine
(SAM). It had been independently described for a handful of enzymes before the enzymes were not only found to
share some features [2,3], but a bioinformatics study
elevated this ragtag group of individuals to an armada
of over 600 putatively related enzymes, appropriately
denoted ‘Radical SAM’ enzymes [4]. Understanding of
individual family members has correspondingly been
greatly enhanced, providing new impetus to investigate
their diverse functions in numerous branches of metabolic pathways.
The first published crystal structure of a Radical SAM
enzyme is that of HemN, the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli [5].
HemN catalyses the prepenultimate step in anaerobic
heme biosynthesis, the conversion of coproporphyrinogen III to protoporphyrinogen IX (Figure 1a). The moderately high resolution of 2.1 Å allows the location and
coordination of cofactors to be described with high precision. Shortly thereafter the crystal structure of biotin
synthase (BioB) also from E. coli was published [6]. BioB
catalyses the last step in biotin biosynthesis, the insertion
of a sulfur atom into the precursor dethiobiotin (Figure
1b). The implication of these structures will be discussed
after briefly reviewing Radical SAM enzymes in general.
Several excellent reviews on Radical SAM enzymes
[2,3,7,8,9] cover aspects only touched upon in the
following.
Radical SAM enzymes
Radical SAM enzymes are found in all three kingdoms of
life and participate in numerous biosynthetic pathways.
Founding family members include lysine 2,3-aminomutase (LAM), lipoate synthase (LipA), BioB, spore
photoproduct lyase (SPP lyase) as well as the activating enzymes of anaerobic ribonucleotide reductase
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Structure and function of radical SAM enzymes Layer et al. 469
Figure 1
(a)
–O
2C
M
M
A
–O
CO2–
B
NH
HN
NH
HN
C
D
M
M
M
4 e–
2 CO2
CO2–
2C
B
A
NH
HN
NH
HN
C
D
M
–O
Coproporphyrinogen III
(b)
Heme
M
M
Chlorophyll
CO2–
2C
Protoporphyrinogen IX
O
O
‘S’
HN
9
H3C
NH
6
H2C
HN
BioB
CO2–
Dethiobiotin
9
NH
6
CO2–
S
Biotin
Current Opinion in Chemical Biology
HemN- and BioB-catalyzed reactions. (a) The overall reaction scheme of coproporphyrinogen III oxidases (CPO). Under anaerobic conditions,
HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX by consecutively oxidizing two propionate
side chains of rings A and B to the corresponding vinyl groups. (b) An overview of the reaction through which biotin synthase (BioB) inserts a
sulfur into dethiobiotin producing biotin.
(aRNR-AE), pyruvate formate-lyase (PFL-AE) and benzylsuccinate synthase (BssD-AE) [8,9]. More recently
characterized members include HemN [5,10]; MiaB, a
tRNA-methylthiotransferase [11]; ThiH, involved in
thiamine biosynthesis [12]; 7,8-didemethyl-8-hydroxy5-deazariboflavin synthase [13]; AtsB [14]; DhaB2, the
B12-independent glycerol dehydratase activating enzyme
[15]; and HydE/G, required for the maturation of an
active [Fe]-hydrogenase [16].
Common features
Despite their surprisingly diverse functions, Radical SAM
enzymes share some common features: all contain an
unconventional [4Fe-4S] cluster coordinated by three
rather than four closely-spaced cysteine residues, creating
the defining CxxxCxxC motif of this family [3,10,18–19].
Thoroughly studied clusters include those of LAM
[20,21], BioB [22,23], LipA [24], aRNR-AE [19] and
PFL-AE [25]. [4Fe-4S] centers of Radical SAM enzymes
are very labile, easily decomposing to [3Fe-4S] or [2Fe2S] clusters [19,26–30].
Radical SAM enzymes, furthermore, require the cofactor/
co-substrate SAM to be placed in immediate vicinity of
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the [4Fe-4S] cluster to directly coordinate the cluster and
allow electron transfer from one to the other [8]. The
reaction steps common to all Radical SAM enzymes are
illustrated in Figure 2.
Reduction of the iron-sulfur cluster
The first common step in all Radical SAM enzyme reactions is the reduction of the [4Fe-4S] centers from the
resting +2 to the active +1 state (Figure 2). Reduced
flavodoxin is the electron donor for some Radical SAM
enzymes in E. coli [31–34], for plant BioB it is adrenodoxin [35].
The reduced iron–sulfur cluster initiates SAM cleavage
The reduced [4Fe-4S] center ([4Fe-4S]+) [17,36,37] is
believed to transfer an electron to the sulfonium of SAM,
resulting in its homolytic cleavage to methionine and the
highly reactive 50 -deoxyadenosyl radical (Figure 2).
Methionine and 50 -deoxyadenosine are correspondingly
produced by several Radical SAM enzymes [17,38–41]
but the 50 -deoxyadenosyl radical is too reactive to be
observed directly. Nevertheless, an allylic analogue of
this radical has been observed, supporting its involvement
in the reaction [42,43].
Current Opinion in Chemical Biology 2004, 8:468–476
470 Mechanisms
Figure 2
HOOC
NH2
+
Flavodoxinox
[4Fe-4S] 1+
Flavodoxinred
[4Fe-4S]2+
H3C S
CH2
Ado
SAM +
HOOC
NH2
+
•
CH2
R-H
Ado
H3C S
Methionine
CH3
Ado
R•
5′-Deoxyadenosine
Current Opinion in Chemical Biology
Reaction steps common to all Radical SAM enzymes. First, an external electron donor (usually flavodoxin) reduces the [4Fe-4S] center to the +1
state. Second, the [4Fe-4S]1+ center transfers an electron to the sulfonium of SAM causing the homolytic cleavage of SAM to methionine and a
highly reactive 50 -deoxyadenosyl radical. Third, the 50 -deoxyadenosyl radical abstracts a hydrogen from an appropriately placed substrate (R–H),
creating a substrate-based radical (R).
Hydrogen atom abstraction
The 50 -deoxyadenosyl radical, resulting from the reductive cleavage of SAM, abstracts a hydrogen atom from an
appropriately positioned carbon. If the source of this
hydrogen is an organic substrate molecule giving rise to
the corresponding substrate radical, the Radical SAM
enzymes are true enzymes (e.g. LAM, BioB, SPP lyase,
HemN, LipA, MiaB). Alternatively, the substrate may be
a protein, generating a catalytic glycyl radical. Such
Radical SAM enzymes function as activases (e.g.
ARNR-AE, PFL-AE, BssD-AE, DhaB2). Glycyl radicals
on proteins are stable and may be observed by electron
paramagnetic resonance (EPR) [44–46]. Substrate radicals, by contrast, are usually more reactive. The only
Radical SAM enzyme for which substrate radicals have
been observed directly is LAM [47,48]. In other cases,
incorporation of a substrate H-atom into 50 -deoxyadenosine indirectly demonstrates the involvement of substrate
radicals [49,50].
Reaction steps following H-atom abstraction are unique
to each enzyme. In some cases, the product radical
intermediate re-abstracts a hydrogen from 50 -deoxyadenosine, restoring SAM, which therefore acts as a true
cofactor. Mostly, however, SAM is consumed and functions as a co-substrate.
Cofactor geometry of Radical SAM enzymes
The reductive cleavage of SAM mediated by the
reduced [4Fe-4S] cluster in Radical SAM enzymes
necessitates a close interaction of the cofactors. This
Current Opinion in Chemical Biology 2004, 8:468–476
interaction has been investigated for LAM, PFL-AE
and BioB using Mössbauer, EPR, resonance Raman and
electron nuclear double resonance (ENDOR) spectroscopy. Se X-ray absorption spectroscopy experiments for
LAM indicated a distance of 2.7 Å between Se of SeMet (the cleavage product of Se-SAM) and an iron atom
of the cluster — the first indirect spectroscopic evidence
for SAM binding to the [4Fe-4S] cluster [51]. An equivalent interaction could not be shown for PFL-AE and
BioB, possibly because SAM is a co-substrate in PFLAE and BioB whereas it serves as a cofactor in LAM [52].
Mössbauer studies of PFL-AE [53] and BioB [54] indicate that a unique non-cysteine-ligated Fe in the cluster
of Radical SAM enzymes is coordinated by SAM
instead. ENDOR spectroscopic studies of PFL-AE with
2
H- and 13C-labelled SAM [55] and with 17O- and 15Nlabelled SAM [56] — repeated for LAM [57] —
revealed that SAM coordinates the unique Fe through
the amino nitrogen and one carboxylate oxygen of the
methionine moiety of SAM. The methyl carbon and the
closest methyl proton of SAM were estimated to be
about 4–5 Å and 3 Å, respectively, from the nearest iron
atom [55]. In the derived model, the methionine moiety of SAM forms a five-membered-ring chelate to the
unique iron atom of the cluster and the SAM sulfonium
interacts with a sulfide of the cluster [56] (Figure 3a).
Overall, the binding mode was the same for PFL-AE
and LAM, although slight differences in the binding
geometry probably reflect the different roles of SAM
during catalysis (co-substrate for PFL-AE, cofactor for
LAM) [57].
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Structure and function of radical SAM enzymes Layer et al. 471
Figure 3
HemN
(a)
H
H
Ado
H
Cysteine
3–3.8 Å
S+
4–5 Å
Cysteine
Fe
S
S
Fe
H2
N
S
Fe
Fe
S
Cysteine
O
O
(b)
C69
C66
2.4
3.7
3.5
2.5
2.3
2.6
2.4
C62
Fe
4.3
M
Current Opinion in Chemical Biology
Derived structures of the [4Fe-4S] center and SAM. (a) Spectroscopic
studies indicate that the unique iron of the [4Fe-4S] center is
coordinated by the amino group nitrogen and a carboxylate oxygen of
the methionine moiety. ENDOR experiments furthermore assign a
distance of 3–3.8 Å between one of the SAM methyl hydrogen atoms
and the nearest iron. This translates into a distance of 4–5 Å from
this iron to the methyl carbon [55,56]. (b) The relative orientations
of the [4Fe-4S] cluster, the coordinating cysteines and SAM as
refined in the structure of HemN. Note the good agreement
between the ENDOR and X-ray structurally derived distances between
the unique Fe of the cluster and the SAM methyl-group (M).
Crystal structures of Radical SAM enzymes
The first two crystal structures of Radical SAM enzymes
have recently been published, those of HemN [5] and
BioB [6]. They confirm many of the results obtained for
these and other Radical SAM enzymes in particular as
concerns the geometry of the cofactors and the relevance
of the [4Fe-4S] center binding motif. The structures
reveal some similarities that are discussed and compared
in the following.
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Tetrapyrroles, such as hemes and chlorophylls, are essential cofactors for numerous enzymes in most organisms.
Their biosynthesis requires the coordinated activity of
highly diverse enzymes [58,59]. Following cyclization of
four pyrroles and decarboxylation of four acetyl to methyl
groups, the propionate side chains on rings A and B of the
intermediate coproporphyrinogen III are oxidatively decarboxylated to the corresponding vinyl groups, yielding
protoporphyrinogen IX [59] (Figure 1a). This enzyme
activity is catalyzed by two coproporphyrinogen III oxidases (CPOs): HemF (oxygen-dependent CPO) [60] and
HemN [10]. Oxygen-dependent HemF participates in
aerobic heme biosynthesis, whereas oxygen-independent
HemN is utilized in anaerobic heme biosynthesis. Oxygen-independent CPO activity was first reported in cellfree extracts under anaerobic conditions in 1969 [61]. The
first bacterial hemN gene putatively expressing an anaerobic coproporphyrinogen III oxidase was sequenced in
1992 [62].
The postulated membership of HemN in the Radical
SAM protein family [4] has been confirmed through
biochemical characterization of recombinant HemN from
Escherichia coli [10]: initial steps in the reaction of HemN
follow the pattern of other Radical SAM enzymes,
described above. The physiological electron donor of
HemN for the initial reduction of the [4Fe-4S] center
remains to be identified. In vitro, NAD(P)H can substitute for the initial electron source [10], indicating that
flavodoxin could also serve as reductant in this reaction.
The presence of a [4Fe-4S] center is indicated by the UV/
vis-absorption characteristics [10] and more recently by
other spectroscopic methods (G Layer, D Jahn, unpublished results). SAM has been shown to be mandatory for
HemN activity [10], and methionine formation during
the reaction was recently observed (G Layer, D Jahn,
unpublished results).
Following reductive cleavage of SAM, the reaction of
HemN deviates from that of other Radical SAM
enzymes. The postulated mechanism for the HemN
reaction involves the stereospecific hydrogen abstraction of the pro-S hydrogen from the propionate side
chain b-C of coproporphyrinogen III [10]. Stereospecific loss of this hydrogen had been shown in early studies
using cell-free extracts [63]. The mechanism indicates
the involvement of a coproporphyrinogenyl radical in
this reaction, which we have been able to verify spectroscopically (unpublished results). The final reaction step
in the HemN-catalyzed reaction is the decarboxylation
of the coproporphyrinogenyl III radical releasing CO2
and concomitant formation of the vinyl group (Figure 4).
The remaining lone electron must be transferred to
an electron acceptor, which remains to be identified.
The described reaction needs to be repeated for the
second propionate side chain of coproporphyrinogen III,
Current Opinion in Chemical Biology 2004, 8:468–476
472 Mechanisms
Figure 4
–OOC
–OOC
α
•
H2C
Ado
H
+
α
β
+
R
Ado
β
e–
• β
H2CH
M
α
Electron acceptor
R
M
R
M
CO2
NH
NH
R
R
R
Coproporphyrinogen III
NH
Coproporphyrinogenyl III radical
Protoporphyrinogen IX
Current Opinion in Chemical Biology
The proposed reaction mechanism of HemN. Following reductive cleavage of SAM, the resulting 50 -deoxadenosyl radical (Ado-CH2) abstracts
a hydrogen atom from the pyrrole propionate Cb-atom. This induces side-chain decarboxylation but requires the resulting radical to be
quenched by a terminal electron acceptor.
requiring a second SAM molecule to bind to the active
site.
Structure of HemN
The crystal structure of HemN (Figure 5) revealed a
monomeric, two-domain enzyme consisting of the catalytic N- (shown in blue) and an a-helical C-terminal
domain (shown in magenta). The N-terminal 30 residues
create an extended ‘trip-wire’ (shown in green) that runs
along a groove created by the N- and C-terminal domains.
Structurally, both trip-wire and C-terminal domain appear
to participate in substrate binding and active-site closure
[5]. The catalytic domain is dominated by a curved, 12stranded, largely parallel b-sheet centered around six
consecutive b-strand/a-helix (b/a)-motifs that group
around a central axis reminiscent of (b/a)8- or TIM-barrel
domains. Apart from the missing two (b/a)-units, the
curvature especially as regards the first and last unit is
wider than in (b/a)8-barrels, such that two additional
units would not complete the barrel. At either side of
the laterally opened barrel, b-sheets extend the barrel
b-sheet, creating an V-shaped domain. This is closed
from above by the conserved cysteine loop (see below)
and from below by two short b-strands, leaving a lateral
substrate-binding site.
Unexpectedly, HemN binds three rather than two cofactors [5], roughly arranged along the b-barrel axis. The
[4Fe-4S] cluster is located uppermost (dark green cube,
Figure 5a,d), protected from above by a flattened polypeptide loop that connects the first b-strand to the following a-helix. This loop bears the conserved cysteine
residues (shown as yellow spheres) that combine to create
the CxxxCxxC motif characteristic of all Radical SAM
enzymes. Each cysteine coordinates an iron of the [4Fe4S] cluster. Just below the [4Fe-4S] cluster, a SAM
molecule (shown in red) is positioned in such a way that
its amino-group nitrogen and one oxygen of its carboxCurrent Opinion in Chemical Biology 2004, 8:468–476
ylate group coordinate the unique fourth iron of the [4Fe4S] cluster. Below the first SAM but slightly offset from
the b-barrel axis, a second SAM (SAM2) has been identified. As HemN successively catalyzes two propionate
side-chain decarboxylations per substrate molecule,
SAM2 could be functionally relevant especially as the
distance between the propionate side chains could
thereby be bridged, circumventing the need to release
a harderoporphyrinogen intermediate. Further biochemical characterization will, however, be required to corroborate this interpretation.
Structure and function of biotin synthase
BioB catalyzes the final step in the multistep biosynthesis
of the vitamin biotin in bacteria, plants and mammals
[64]. The reaction is unusual in that two non-activated
hydrogen atoms need to be abstracted to introduce two
S–C bonds to C6 and C9 (Figure 1b).
The 3.4 Å resolution crystal structure of BioB from E. coli
confirms that this is a homodimeric, single-domain protein (Figure 5b) [6]. Structurally, BioB clearly falls into
the (b/a)8- or TIM-barrel family (blue), with an additional
a-helical N-terminal extension of ~30 residues (green).
The structure reveals the location of all cofactors, a [4Fe4S] cluster, SAM and a [2Fe-2S] cluster, as well as the
substrate dethiobiotin. All cofactors are positioned near
the central b-barrel axis. The [4Fe-4S] cluster is uppermost (green cube in Figure 5b/pale green in 5c and 5d),
coordinated by the three conserved cysteines of the
Radical SAM CxxxCxxC motif. SAM (red/white) is
located immediately below, coordinating the fourth iron
of the cluster and a [2Fe-2S] (black/light grey) is located
in the lower half of the b-barrel. The substrate dethiobiotin (orange/pale orange) is located intermediate
between SAM and the [2Fe-2S], indicating that this
cluster may be the sacrificial donor of the sulfur that is
incorporated to produce biotin [6,65,66].
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Structure and function of radical SAM enzymes Layer et al. 473
Figure 5
(a)
(b)
N
N
BioB
C
HemN
C
(c)
α4
α3
(d)
α2
HemN
BioB
4Fe-4S
SAM
β4
α5
β3
β2
β1
β5
β6
α1
SAM2/
DTB
2Fe-2S
Current Opinion in Chemical Biology
Crystal structures of HemN and BioB. (a) The crystal structure of the HemN monomer consists of an N-terminal, (b/a) catalytic domain
(shown in blue), an a-helical C-terminal domain (magenta) and an N-terminal extension of the catalytic domain without significant secondary
structure (green). The protein binds a [4Fe-4S] cluster (green) and SAM (red), the latter coordinating the unique fourth iron of the iron–sulfur
cluster not bound by a cysteine. Both cofactors/coreactants roughly lie along the central b-barrel axis. HemN also binds a second SAM (orange) the
function of which is not entirely clear. (b) The crystal structure of BioB. Structurally, each monomer of the homodimeric BioB belongs to the (b/a)8- or
TIM-barrel fold (blue) with an a-helical, N-terminal extension (green). BioB binds three cofactors/coenzymes roughly along the central barrel
axis. Uppermost is the [4Fe-4S] center (green) coordinated by three conserved cysteines (yellow spheres) and an immediately neighboring
SAM (red) lying just below the iron–sulfur center. Roughly half-way along the barrel axis, a [2Fe-2S] cluster (grey) completes the arrangement of
cofactors. Between the SAM and the [2Fe-2S] center but slightly offset from the barrel axis, BioB harbors the substrate dethiobiotin (orange).
(c) A structural comparison of HemN (colored) and BioB (pale colors). Only those parts of the structures delimited by barrel b-strands 1 to 6
and the cofactors are shown. Note the similarity of b-strands 3 to 6, of the neighboring a-helices and of the cofactors: the [4Fe-4S] cluster (green,
pale green) and SAM (red, pink). The location of dethiobiotin (pale orange) is similar to that of SAM2 (orange). (d) A structural superposition of
cofactors, substrates and the CxxxCxxC [4Fe-4S]-coordinating loop. Detailed differences especially as regards the [4Fe-4S] cluster (green, pale
green) and SAM (red, pink) may either be due to differences in the proteins or to the fact that BioB has been refined at a lower resolution (3.4 Å)
compared with HemN (2.1 Å), resulting in inherently more accurate atomic positions in HemN.
Comparison of HemN and BioB protein
structures
Structurally, HemN and BioB may initially appear surprisingly different as HemN consists of two distinct
domains and BioB of only one (Figure 5a,b). Closer
inspection, however, reveals strong similarities. Each
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catalytic domain is related to a (b/a)8- or TIM-barrel,
although only a subset of four neighboring (b/a)-motifs
truly constitute the cofactor-binding domain, which
accommodates a linear arrangement of cofactors within
its central curved b-sheet. Iterative superposition indicates that 90 residues of both structures may be matched
Current Opinion in Chemical Biology 2004, 8:468–476
474 Mechanisms
with interatomic distances of mainchain atoms of <3 Å
and a root-mean-square deviation of 1.6 Å. The region
immediately surrounding the cofactors is most strongly
conserved (b-strands 3, 4, 5 and 6) and as the cofactors
generally lie near the C-terminal end of the parallel bstrands, this side of the structure is most similar (Figure
5c). The loop connecting b-strand 1 and the following ahelix bears the conserved cysteines of the CxxxCxxC
motif and is conformationally similar in both structures,
especially so for the terminal xxCxxC region. Surprisingly, the residue following the first cysteine is a proline
in BioB and a histidine in HemN, resulting in a significant
deviation in the position of the first cysteine (right-hand
yellow sphere in Figure 5c).
Similarities in cofactor arrangements
The arrangement of the cofactors is similarly analogous in
both HemN and BioB (Figure 5d). The [4Fe-4S] cluster
is located uppermost near the protein surface, bound by
three cysteines of the conserved cysteine-bearing loop.
The relative orientation and the centroid positions coincide remarkably well despite the lower precision afforded
for by the medium resolution of the BioB structure. This
difference is reflected by the overall shape of the cluster,
which is cubic in BioB rather than distorted cubic as
observed in HemN and other high-resolution crystal
structures.
Immediately below the [4Fe-4S] cluster along the bbarrel axis, the catalytic SAM molecule is arranged to
coordinate the fourth, unique iron of the cluster not
ligated by cysteine. Again the positional overlap of the
two structures clearly indicates that the geometry of
ligation is roughly identical despite significant pair-wise
differences (Figure 5d) presumably due to resolutionlimited modeling in BioB. In principle, both structures,
nevertheless, confirm the ENDOR-derived model that
had indicated iron chelation by the amino nitrogen and
one carboxylate oxygen of the methionine moiety (Figure
3a) [56]. Note in particular the excellent agreement
between HemN (4.2 Å) and the ENDOR (4–5 Å) model
in the distance between iron and the methyl group of
SAM (Figure 3b) — even though the iron involved is the
SAM-ligated iron and not one of the cysteine-coordinated
ones.
Surprisingly the position of dethiobiotin in BioB closely
matches that of the SAM2 in HemN (pale orange/orange
in Figure 5d). The significance of this overlap is unclear,
especially due to the lack of functional similarity —
dethiobiotin is the substrate of BioB whereas SAM2 is
a proposed cofactor of HemN. Also, although the [2Fe2S] cluster of BioB is without counterpart in HemN, its
position exactly matches a water-filled space in HemN
that has been proposed to accommodate the electron
acceptor [5], required for the final step in the decarboxylation of coproporphyrinogen III.
Current Opinion in Chemical Biology 2004, 8:468–476
Overall, the binding pocket of the SAM adenine moiety
is amongst the most conserved elements between HemN
and BioB. In particular, the loop Ile211-Ile212-Gly213Leu214 (HemN) is conformationally identical to the loop
Ile192-Val193-Gly194-Leu195 (BioB). Similarly, loop
Phe240-Ala243 (HemN) is equivalent to Asn222Val225 (BioB), the last residue coordinating the N1
and N6 of the SAM adenine ring via its backbone nitrogen
and carboxylate oxygen. Of the three motifs proposed to
be conserved in Radical SAM enzymes [4] only the
CxxxCxxC motif has an obvious function in HemN
and BioB. The remaining homologies appear coincidental or of marginal importance. A surprising difference of
HemN and BioB is the recognition specificity of the
methionine and ribose moieties in the two structures.
Whereas the carboxylate group is positioned precisely
and bound tightly through a bidentate salt bridge by
Arg184 [5] in HemN, it is coordinated much more
loosely by Arg173 in BioB. Also, essentially every oxygen
or nitrogen atom in SAM of HemN is either a hydrogenbond donor or acceptor, whereas this is frequently not the
case in BioB.
Conclusions
The structural characterization of two members of the
Radical SAM family has brought a degree of maturity to
this field of research. Their partial homology confirms the
classification of Radical SAM enzymes as a common
structural family, as variously proposed in the past.
Nevertheless, marked structural differences between
HemN and BioB are just as clear, indicating that the
protein core has been adapted to a unique task, yielding at
least two sub-classes in the process, a (b/a)8-barrel group
as exemplified by BioB and a class bearing an opened (b/
a)6-core as observed in HemN. Pertinent details of the
structures had been inferred by spectroscopic and biochemical analyses, many of which have been vindicated
by the structural data. Specific questions on individual
family members may now be addressed working from a
firm structural foundation, describing both the protein
backbone and the cofactors embedded within.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft
(DWH and DJ).
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