Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes
2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês
10.1093/emboj/cdg598
ISSN1460-2075
Autores Tópico(s)Advanced battery technologies research
ResumoArticle1 December 2003free access Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes Gunhild Layer Gunhild Layer Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Jürgen Moser Jürgen Moser Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Dirk W. Heinz Dirk W. Heinz Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38104 Braunschweig, Germany Search for more papers by this author Dieter Jahn Dieter Jahn Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Wolf-Dieter Schubert Corresponding Author Wolf-Dieter Schubert Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38104 Braunschweig, Germany Search for more papers by this author Gunhild Layer Gunhild Layer Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Jürgen Moser Jürgen Moser Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Dirk W. Heinz Dirk W. Heinz Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38104 Braunschweig, Germany Search for more papers by this author Dieter Jahn Dieter Jahn Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany Search for more papers by this author Wolf-Dieter Schubert Corresponding Author Wolf-Dieter Schubert Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38104 Braunschweig, Germany Search for more papers by this author Author Information Gunhild Layer1, Jürgen Moser1, Dirk W. Heinz2, Dieter Jahn1 and Wolf-Dieter Schubert 2 1Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany 2Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38104 Braunschweig, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6214-6224https://doi.org/10.1093/emboj/cdg598 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info ‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 Å resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 Å) and a neighboring sulfur of the cluster (3.6 Å), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme. Introduction Biosynthesis of heme and chlorophyll requires coproporphyrinogen III to be converted to protoporphyrinogen IX by oxidatively decarboxylating the propionate side chains of rings A and B to the corresponding vinyl groups (Figure 1A). Two unrelated enzymes catalyze this reaction: HemF, the oxygen-dependent coproporphyrinogen III oxidase; and HemN, the oxygen-independent coproporphyrinogen III oxidase (Jordan, 1981; Dailey, 2002; Friedmann and Tauer, 1992; Chadwick and Ackrill, 1994). Figure 1.Schematic representation of the enzymatic reaction of HemN. (A) HemN oxidatively decarboxylates coproporphyrinogen III to protoporphyrinogen IX by converting the propionate side chains of rings A and B to the corresponding vinyl groups. (B) The first reaction step common to HemN and all Radical SAM enzymes: a reduced 4Fe–4S cluster transfers an electron to the sulfonium of S-adenosylmethionine (SAM). The C5′–S+ bond of SAM is cleaved, producing methionine and a highly oxidizing 5′-deoxyadenosyl radical. The radical abstracts a hydrogen atom from a substrate RH (the substrate may itself be an enzyme), creating the corresponding substrate radical (R·). (C) In the reaction catalyzed by HemN, the 5′-deoxyadenosyl radical abstracts a hydrogen atom from the β-C atom of the substrate propionate side chain. CO2 is eliminated, and a single electron transfer to an electron acceptor gives rise to the vinyl group of the reaction product. Download figure Download PowerPoint The oxygen-independent coproporphyrinogen III oxidase HemN, an iron–sulfur protein, belongs to the recently discovered ‘Radical SAM’ protein family (Sofia et al., 2001; Layer et al, 2002). All members of this protein family contain an unusual 4Fe–4S cluster coordinated through three conserved cysteine residues in a characteristic CxxxCxxC motif (Sofia et al., 2001). In its reduced state, the iron–sulfur cluster transfers a single electron to S-adenosylmethionine (SAM), inducing the reductive cleavage of SAM to methionine and a 5′-deoxyadenosyl radical. This highly oxidizing radical abstracts a hydrogen atom from an appropriately positioned carbon atom, creating a substrate (as in HemN, lysine-2,3-aminomutase) or a catalytic glycyl radical [as in activating enzymes of class III ribonucleotide reductase and pyruvate formate-lyase (Cheek and Broderick, 2001; Fontecave et al., 2001; Frey and Magnusson, 2003; Jarrett, 2003) (Figure 1B). Whereas SAM is consumed in some Radical SAM enzymes [HemN, biotin synthase (Uglava et al., 2003) and pyruvate formate lyase-activating enzyme (Frey et al., 1994)], it is restored and reused in others [spore photoproduct lyase (Cheek and Broderick, 2002) and lysine-2,3-aminomutase (Frey and Magnusson, 2003)]. Another feature common to all Radical SAM proteins is a glycine-rich sequence motif proposed to be the SAM-binding site. Radical SAM enzymes are found in numerous fundamental biosynthetic pathways such as vitamin, cofactor, DNA precursor or antibiotic biosyntheses. Despite catalyzing widely different reactions and having very low sequence conservation, Radical SAM enzymes are presumed to bear a common core domain (Sofia et al., 2001). Three-dimensional information has, however, been lacking so far, despite a wealth of biochemical data for many members of the family. We present the crystal structure of HemN, the first structure determination of a member of the Radical SAM family. The iron–sulfur protein was co-crystallized with the cofactor SAM. The 2.07 Å crystal structure provides insight into two SAM-binding sites, one of these in close proximity to the iron–sulfur cluster revealing the molecular basis of the first common reaction step of all Radical SAM enzymes. Results Structure determination and refinement HemN was expressed, purified and crystallized under strictly anaerobic conditions in an anaerobic chamber. This ensured the integrity of the 4Fe–4S cluster in a large proportion of the enzyme population, stabilizing the molecular structure and allowing successful protein crystallization. Diffraction data of cryo-cooled crystals were collected at the Fe K-edge and the crystal structure was solved by multiple anomalous difference (MAD) techniques (Table I). The anomalous signal extends to the resolution limit, allowing the localization of the iron–sulfur cluster by Patterson methods. AUTOSHARP (http://www.globalphasing.com) was used for integrated phasing (SHARP; de la Fortelle and Bricogne, 1997), solvent flattening (DM; Cowtan and Main, 1998) as well as automated model building (ARPWARP; Lamzin and Wilson, 1993) and refinement (REFMAC5; Murshudov et al., 1997), greatly enhancing the speed of structure solution. Due to severe radiation sensitivity, the 1.80 Å high-resolution data set collected after the MAD experiment proved ineffectual in phasing and of limited use in refinement, as disordered regions are significantly extended. The structure of HemN has therefore been refined against the first data set. The final R-factor is 15.4% (Rfree = 18.7%) with good geometry (Table I). Overall, 439 of 457 residues have been located in the electron density map. Disordered regions of the polypeptide not included in the final model include the residues 1–3, 19–21 and 446–457. Residues 4–6, 17–18 and 22–24 are partly disordered but have been included in the final model. Three residues lie outside the favored regions of the Ramachandran plot: Ser25 in the poorly defined N-terminal strand and Met287 at the interface between the catalytic domain and the N-terminus are both partly disordered and presumably adopt an ordered conformation only on substrate binding. Gln172 is involved in SAM binding. Crystal contacts exclusively involve the catalytic domain of HemN. Modeling interdomain movement during refinement (TLS refinement; Murshudov et al., 1997) indicates significant freedom of movement of the C-terminal domain and N-terminal trip-wire (see below). Correspondingly, the average temperature factor of these domains is 50.4 Å2, in contrast to 25.4 Å2 for the catalytic domain (including cofactors). Table 1. Data collection and refinement statistics Data collection Data set Inflection Peak High energy remote Wavelength (Å) 1.742 1.739 1.542 Space group P63 Unit cell lengths (Å) a = b = 114.0, c = 76.5 Resolution range (Å)a 30−2.07 (2.11−2.07) 30−2.06 (1.53−1.50) 30−1.84 (1.86−1.84) Rmerge (%)a 6.6 (18.2) 7.4 (14.4) 7.7 (38.5) I/σIa 30.6 (10.2) 29.2 (9.7) 27.0 (3.4) Completeness (%)a 99.1 (96.8) 97.4 (90.7) 98.8 (84.3) Redundancya 9.4 (8.8) 8.5 (2.7) 8.5 (4.5) Unique reflections 34 220 34 613 48 413 Wilson plot B-factor 27.2 26.0 25.5 Refinement Resolutiona 20−2.07 (2.12−2.07) R (%)a 15.4 (15.8) Rfree (%)a 18.7 (21.5) No. of reflectionsa Working set 59 440 (2334) Test set 3170 (133) Water molecules 398 Average B-factor (Å2)b 34.0 (15.8) R.m.s.d. bond lengths (Å) 0.024 R.m.s.d. bond angles (°) 2.1 Ramachandran plot (%) Allowed 92.3 Additional 7.2 Generous 0 Disallowed (%) 0.5 a Values in parentheses correspond to the highest resolution shell. b The value in parentheses indicates the B-factor after TLS refinement. Overall structure of HemN HemN is a monomeric protein consisting of two distinct domains (Figure 2A). The N-terminal domain, significantly larger than the C-terminal domain, comprises residues 36–364. It is characterized by a curved, 12-stranded, largely parallel β-sheet. Only three of the 12 strands, located near or at the very end of the sheet, arrange in an antiparallel fashion (Figure 2B). α-Helices predominantly decorate the outer surface of the β-sheet. The six N-terminal β-strands (residues 50–282) are part of repeated β/α motifs (or βαα variations) forming the central core of the domain. Structurally, the curved (β/α)6 repeat bears some resemblance to known (β/α)8 or TIM barrel domains, including that of the β-amylase of Bacillus cereus (PDB code: 1B9Z), human glucuronidase (1BHG) and, intriguingly, uroporphyrinogen decarboxylase (1URO) or HemE, the preceding enzyme in heme biosynthesis (Whitby et al., 1998). Compared with the TIM barrel, individual β-strands are, however, less strongly inclined relative to the barrel axis and the curvature of the β-sheet is not nearly as tight. The missing (β/α) motifs of the (β/α)6 or three-quarter barrel open the barrel laterally, resulting in a substrate-binding pocket perpendicular to the β-barrel axis rather than aligned with this axis as in many TIM barrel proteins. Similarly to TIM barrels, loops at the N-terminal ends of β-strands tend to be short while those at the C-terminal ends are often long, without much additional secondary structure. This is particularly true for the two loops following strands β1 and β6, which cover the central void of the β-barrel on the C-terminal side, while a single long loop (after β-strand 8) bearing two short β-strands connected by a hairpin loop (b1–loop–b2 in Figures 2B and 3B) plugs the void on the opposite side, leaving only the lateral opening. Figure 2.Structure of HemN. (A) A ribbons-type and (B) a schematic representation of the secondary structure elements. HemN consists of two distinct domains (shades of blue and red) as well as an elongated N-terminal region termed a trip-wire (green). The catalytic domain is built around a 12-stranded, largely parallel β-sheet. At its core, the N-terminal region bears a three-quarter barrel, a (βα)6 variation of the (βα)8 TIM barrel. This core binds all cofactors, a 4Fe–4S cluster and two SAM molecules. The N-terminal trip-wire and the C-terminal domain probably participate in substrate binding. A CxxxCxxC motif, conserved in all Radical SAM proteins, is located in a loop following the first β-strand of the central barrel. The three cysteines (small yellow circles) bind three of the Fe ions of the cluster. Download figure Download PowerPoint Figure 3.Detailed views of the cofactors. (A) The electron density associated with the cofactors and the CxxxCxxC motif, conserved in all Radical SAM proteins. The 4Fe–4S cluster is rendered in green (Fe) and yellow (S), while pink-colored bonds highlight SAM1 and SAM2. Both (S)- (above) and (R)- (below) sulfonium sulfur configurations are observed for SAM1. SAM2 is rotationally disordered around the C5′–S+ bond, resulting in discontinuous electron density. (B) The cofactors occupy the central void of the catalytic domain near the C-terminal ends of the three-quarter barrel β-strands. Orange spheres mark the Cα positions of conserved cysteines. (C) A schematic depiction of inter-cofactor distances and amino acid residues involved in binding the cofactors. The (S)-sulfur is presented in yellow and the (R)-sulfur in orange. Green arcs represent hydrophobic interactions. Download figure Download PowerPoint The β-sheet of the three-quarter barrel is extended at either end by additional β-strands deepening the substrate-binding tunnel. Though physically located in the middle of the domain, the three-quarter barrel corresponds to the N-terminal residues 50–282. At its C-terminal end, it is directly complemented by two β-strands followed by a long loop bearing the β-strand–loop–β-strand plug of the central void. The polypeptide then leads back to the N-terminal end of the three-quarter barrel, where a further four β-strands complete this domain. The combination of the 12-stranded β-sheet closed above and below the β-barrel creates a domain reminiscent of a cupped hand or crucible with a deep active site tunnel, to accommodate both the cofactors and the large coproporphyrinogen III substrate molecule (see below). As far as we can ascertain, the N-terminal domain is unique. Initial secondary structure prediction analyses of other Radical SAM proteins indicate that the three-quarter barrel with its lateral opening may be a common feature of all Radical SAM proteins. The C-terminal domain connected to the N-terminal domain by a short loop following the last β-strand consists of a bundle of four, roughly parallel, α-helices and a small, three-stranded antiparallel β-sheet. Significant structural homologs have not been identified. However, as this domain bears similarly highly conserved regions as the N-terminal domain, it must be functionally important to the enzyme. Most likely is a role in covering and partly filling the substrate-binding crevice of the N-terminal domain to shield the substrate from the solvent once it has bound. Sequence homology to other Radical SAM proteins could not be detected, indicating that this domain is presumably HemN specific. Structurally, the N-terminal residues (4–35) belong to neither the catalytic nor the C-terminal domain. Instead, the first 35 residues adopt an extended conformation without pronounced secondary structure. It is loosely bound in an extended cleft between the N- and the C-terminal domains, wrapping around the latter. Although partly disordered (residues 19–21), it bears an amino acid sequence G20PRYTSYPTA29 highly conserved in all HemNs possibly involved in substrate recognition, and partly covers the entrance to the active site (see below). Structurally, it may, therefore, function akin to a ‘trip-wire’, stabilizing on substrate binding and possibly inducing the C-terminal domain to rearrange and close the active site. The 4Fe–4S cluster and SAM cofactors The crystal structure of HemN contains three cofactors, a 4Fe–4S cluster and two SAM molecules, henceforth denoted SAM1 and SAM2. The 4Fe–4S cluster and SAM1 are well resolved in the electron density map (Figure 3A). Rotational disorder around the C5′–S+ bond in SAM2 leads to the interruption of the electron density. All cofactors are bound in close mutual proximity within the active site pocket of the catalytic domain near the C-terminal end of the parallel β-strands of the three-quarter barrel (Figure 3B). The 4Fe–4S cluster is bound in the deepest recesses of the active site pocket, near the center of the three-quarter barrel. Three of its Fe ions are coordinated through three characteristically conserved cysteines (Cys62, Cys66 and Cys69 in HemN) of the Radical SAM CxxxCxxC motif (Sofia et al., 2001). The cysteines are located in an extended loop immediately C-terminal of the β-strand β1 (orange spheres in Figure 2). This flattened, circular loop laterally wraps around the 4Fe–4S cluster. Overall, the geometry, individual bonding distances within the cluster and distances to coordinating cysteines are typical of 4Fe–4S clusters. The environment of the cluster is, however, strongly polarized. Hydrophobic residues surround roughly one half of the cluster facing the cysteine-rich loop. Apart from the cysteines, Phe68, which precedes the third conserved cysteine and is invariantly an aromatic residue in all Radical SAM proteins (Sofia et al., 2001), as well as Leu63, Ile74, Val75 and the aliphatic part of Lys73 contribute to this hydrophobic region. Hydrophilic and charged neighbors surround the other half of the cluster. The amide nitrogen of Gly113 and the side chains of Thr114, Asp147, Arg149 and Arg184, and two water molecules are no further than 4 Å from the cluster. An elongated stretch over the fourth Fe, however, remains uncovered by protein. Immediately adjacent to the 4Fe–4S cluster, HemN binds SAM1. The cofactor is well defined and adopts a unique bent conformation. It is held in position by numerous specific interactions (Figure 3C) extensively involving the 4Fe–4S cluster and in turn completing the coordination sphere of the latter: the amino nitrogen and one carboxylate oxygen of the methionine moiety of SAM1 coordinate the fourth iron. The respective distances to the iron are 2.6 (N) and 2.3 Å (O). Like the cysteines coordinating the 4Fe–4S cluster, most residues involved in binding SAM1 are conserved in all HemN sequences (Figure 4A). Arg184 is pivotal in binding the carboxylate group of the SAM methionine moiety, while Q172 adopts a particularly unfavorable backbone conformation to bind both O3′ and O4′ of the SAM ribose moiety. Aromatic residues Phe68, Phe240 and Tyr242 and aliphatic Ile211 surround the adenine moiety, while hydrogen bonds to backbone atoms of Phe68, Gly70 and Ala243 ensure the correct orientation. Surprisingly, the sulfonium sulfur of SAM1 occupies two alternative positions. Both are clearly defined in the anomalous difference maps and are compatible with the expected bond lengths of SAM. The implication is that HemN recognizes SAM with both an (S) and (R) configuration at the chiral sulfonium sulfur. The (S) configuration is favored, as judged by the anomalous difference and electron density maps, representing ∼60% occupancy. In the following, we will refer to (S,S)- and (R,S)-SAM as (S)- and (R)-SAM, as the second chiral center, the methionine Cα atom, invariably bears the (S) configuration. Figure 4.Distribution of conserved residues in HemN. (A) Amino acid sequence of E.coli HemN. Residues conserved in 34 sequences of HemN are underlaid in dark pink; residues conserved in >90, 80 and 70%, respectively, of sequences are marked by progressively lighter shades. α-Helices are represented by rectangles, β-strands by arrows. See Figure 2 for color-coding and nomenclature. Filled circles, squares and inverted triangles below individual residues mark amino acids involved in 4Fe–4S cluster, SAM1 and SAM2 binding, respectively. Filled squares denote residues in domain–domain interactions. Residues postulated to be involved in binding the external electron donor, terminal electron acceptor and the coproporphyrinogen III substrate are marked by half circles, diamonds and plus signs, respectively, Surface representation of HemN (B) front (in stereo) and (C) back view. The degree of conservation (A) is mapped onto the molecular surface of the catalytic domain. Note that the highest concentration of conserved residues is found in the active site cleft and at domain–domain interfaces. The trip-wire and C-terminal domain are represented by green and red coils. Most of the outer surface is poorly conserved (white), with the exception of the proposed entrance to the terminal electron acceptor-binding pocket and, to a lesser extent, the binding site of the external electron donor. Download figure Download PowerPoint In addition to the nitrogen and oxygen ligands to the fourth iron, the sulfonium sulfur of the (S)-SAM stereoisomer is located a mere 3.5 Å from the same iron and 3.6 Å from a neighboring sulfur atom. Presumably, therefore, electron transfer from the reduced 4Fe–4S cluster to SAM occurs through a favorable electronic interaction between the sulfonium sulfur and an Fe–S edge of the cluster. A second binding site for SAM In addition to SAM1, HemN unexpectedly binds a second SAM molecule (SAM2) within the same deep cleft of the N-terminal domain that binds the 4Fe–4S cluster and SAM1. SAM2 is located adjacent to SAM1 and its adenine moiety stacks on the aromatic side chain of Tyr56, conserved in all known sequences of HemN and shown to be essential for HemN catalysis (Layer et al., 2002). A second aromatic residue (Phe310), part of a HemN- conserved 308KNFQGYTT315 motif that creates the strand–turn–strand element plugging the three-quarter barrel (see above), is located on the opposite face of the planar structure. A small change in its χ1 and χ2 torsion angles could swing the side chain to stack upon the opposite face of the adenine moiety. In addition, the ribose moiety is clearly defined in the electron density, while the anomalous difference map indicates the presence of a sulfur covalently attached to the ribose C5′ atom, clearly identifying this molecule as SAM. The electron density is discontinuous after the S, but an additional structure of density is observed adjacent to the S (Figure 3A). We have interpreted this observation as a second molecule of SAM disordered about the C5′–S+ bond. Additional continuous electron density adjacent to the sulfonium sulfur is, however, only incompletely described by the disordered methionine moiety. This may indicate that this site is also partly occupied by a 5′-deoxy-5′-(methylthio)adenosine molecule, a known degradation product of SAM (Hoffman, 1986), plus a second ligand. The identity of this ligand, however, remains unclear. Discussion Radical SAM enzymes SAM is most widely associated with its function of serving as a methyl group donor to methyltransferases, a function that is vital to myriad physiological processes (Schubert et al., 2003). 4Fe–4S clusters are similarly commonly known to function as redox centers in electron transfer reactions. However, in recent years, Radical SAM enzymes have been recognized to combine these two cofactors in a novel fashion. These enzymes bind a 4Fe–4S cluster and SAM in close proximity. Under suitable conditions, reduction of the 4Fe–4S cluster induces electron transfer to the SAM sulfonium, cleaving the C5′–S+ bond to produce the strongly oxidizing 5′-deoxyadenosyl radical. This reactive radical intermediate rapidly abstracts a hydrogen atom from a suitably placed hydrogen donor (protein or substrate) to generate the corresponding radical (Sofia et al, 2001; Frey, 2003; Jarrett, 2003). Structurally, the requirements for a Radical SAM enzyme thus deviate substantially from those of methyltransferases and 4Fe–4S-binding proteins. Methyltrans ferases need to place a methyl group acceptor near the methyl moiety of SAM to facilitate transfer of the methyl group from one to the other. 4Fe–4S clusters are often buried within proteins, allowing electron transfer to occur over fairly large distances while protecting the cluster from direct chemical interaction. Radical SAM enzymes, in contrast, need to pair SAM with a 4Fe–4S cluster in such a way as to allow electron transfer first from an external electron donor onto the 4Fe–4S cluster and then in a second electron transfer step from the reduced cluster to the sulfonium sulfur of SAM. A hydrogen atom donor must be positioned near the ribose C5′ atom preferably prior to the cleavage of the C5′–S+ bond and formation of the 5′-deoxyadenosyl radical to ensure abstraction of the correct H atom. The structure of HemN reveals that the catalytic domain is indeed unique and unrelated to both the typical seven-stranded mixed β-sheet domain observed for SAM-dependent methyltransferases (Schluckebier et al., 1995) and the atypical SAM-binding domains of other methyltransferases (Schubert et al., 2003). It is, furthermore, also unrelated to other 4Fe–4S-binding domains. At its core, the catalytic domain of HemN contains a partial barrel related to the TIM barrel, which we refer to as a three-quarter barrel. The three-quarter barrel bears only six βα motifs opening up the domain laterally. In the case of HemN, the resulting large active site cleft allows the large substrate coproporphyrinogen III to enter the active site. Other features clearly support its role as the functional domain of a Radical SAM protein. The inner parallel β-sheet of the three-quarter barrel covered with α-helices on its outer surface provides overall structural rigidity and stability to the enzyme. The deep lateral active site cleft allows the vital 4Fe–4S cluster to be buried within the protein. This renders it inaccessible to most cell constituents, preventing its strong reduction potential from being lost. The catalytic domain furthermore binds SAM in particular close proximity to the 4Fe–4S cluster. The distances between the cofactors (Figure 3C) are similar to those spectroscopically inferred for pyruvate formate lyase-activating enzyme (Walsby et al, 2002; Cosper et al., 2003), biotin synthase (Cosper et al., 2003) and lysine-2,3-aminomutase (Cosper et al., 2000); however, note the differences below. This further supports the notion that Radical SAM enzymes are closely related. At the same time, structural features of HemN are likely to be conserved in other members of the family. Details that are in agreement include a direct coordination of the fourth Fe ion by both amide nitrogen and one carboxylate oxygen of the SAM methionine moiety. The spectroscopic models differed in their interpretation of whether the sulfonium sulfur of SAM is located nearest the same, fourth iron or an adjacent sulfur atom (Jarrett, 2003). The structure of HemN now reveals that both are, in fact, partly correct as the sulfonium is essentially equidistant from both the fourth iron (3.5 Å) and an adjacent sulfur atom (3.6 Å; Figure 3C). An Fe–C5′ distance of 5.2 Å is, however, nearer the 4.9 Å expected for the S+–S model than the 3.6 Å predicted by the S+–Fe model. Sequence conservation A comparison of 34 amino acid sequences of HemN proteins (Figure 4A) indicates that HemN is remarkably well conserved. Mapping the degree of conservation onto the surface of each domain (catalytic domain shown in Figure 4B) reveals that conserved residues are concentrated in particular areas, most prominently within the active site cleft. Clearly, this correlates with the requirement to bind cofactors and substrate within this cleft. The cysteines coordinating the 4Fe–4S cluster are obviously conserved, as they are conserved in all Radical SAM enzymes (Sofia et al., 2001). Similarly, Phe68, the residue preceding the third conserved cysteine, which was found invariantly to be aromatic in Radical SAM enzymes, is a phenylalanine in ∼50% of HemN sequences and a tyrosine in all others. Located between SAM1 and the 4Fe–4S cluster, this residue contributes to the hydrophobic region of the polarized 4Fe–4S cluster environment (see above) and to binding of the adenine moiety of SAM1 both through van der Waals interactions and through a hydrogen bond
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