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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 www.sciencedirect.com 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 www.sciencedirect.com 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]. www.sciencedirect.com 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. www.sciencedirect.com 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]. www.sciencedirect.com 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 www.sciencedirect.com 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). References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Banerjee R: Radical carbon skeleton rearrangements: Introduction: Radical Enzymology. Chem Rev 2003, 103:2083-2094. This is the introductory article to an issue of Chemical Reviews featuring an excellent collection of reviews on many aspects of radical enzymology. 2. Frey PA: Radical mechanisms of enzymatic catalysis. Annu Rev Biochem 2001, 70:121-148. www.sciencedirect.com Structure and function of radical SAM enzymes Layer et al. 475 3. Cheek J, Broderick JB: Adenosylmethionine-dependent ironsulfur enzymes: versatile clusters in a radical new role. J Biol Inorg Chem 2001, 6:209-226. 4. Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE: 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. Nucleic Acids Res 2001, 29:1097-1106. 5. 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