Title: The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis
Abstract: Article15 January 2003free access The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis Giuliano Sciara Giuliano Sciara Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Steven G. Kendrew Steven G. Kendrew Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Biotica Technology Ltd, 181A Huntingdon Road, Cambridge, CB3 0DJ UK Search for more papers by this author Adriana E. Miele Adriana E. Miele Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Neil G. Marsh Neil G. Marsh Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1055 USA Search for more papers by this author Luca Federici Luca Federici Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Francesco Malatesta Francesco Malatesta Dipartimento di Biologia di Base ed Applicata, Università di L'Aquila, 67100 L'Aquila, Italy Search for more papers by this author Giuliana Schimperna Giuliana Schimperna Istituto G.Donegani, 28100 Novara, Italy Search for more papers by this author Carmelinda Savino Carmelinda Savino CNR, Centro di Studi sulla Biologia Molecolare, c/o Dipartimento di Scienze Biochimiche, Università di Roma ‘La Sapienza’, Piazzale A.Moro, 5, 00185 Roma, Italy Search for more papers by this author Beatrice Vallone Corresponding Author Beatrice Vallone Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Giuliano Sciara Giuliano Sciara Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Steven G. Kendrew Steven G. Kendrew Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Biotica Technology Ltd, 181A Huntingdon Road, Cambridge, CB3 0DJ UK Search for more papers by this author Adriana E. Miele Adriana E. Miele Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Neil G. Marsh Neil G. Marsh Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1055 USA Search for more papers by this author Luca Federici Luca Federici Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Francesco Malatesta Francesco Malatesta Dipartimento di Biologia di Base ed Applicata, Università di L'Aquila, 67100 L'Aquila, Italy Search for more papers by this author Giuliana Schimperna Giuliana Schimperna Istituto G.Donegani, 28100 Novara, Italy Search for more papers by this author Carmelinda Savino Carmelinda Savino CNR, Centro di Studi sulla Biologia Molecolare, c/o Dipartimento di Scienze Biochimiche, Università di Roma ‘La Sapienza’, Piazzale A.Moro, 5, 00185 Roma, Italy Search for more papers by this author Beatrice Vallone Corresponding Author Beatrice Vallone Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy Search for more papers by this author Author Information Giuliano Sciara1, Steven G. Kendrew1,2, Adriana E. Miele1, Neil G. Marsh3, Luca Federici1, Francesco Malatesta4, Giuliana Schimperna5, Carmelinda Savino6 and Beatrice Vallone 1 1Dipartimento di Scienze Biochimiche Università di Roma ‘La Sapienza’, Piazzale A.Moro 5, 00185 Roma, Italy 2Biotica Technology Ltd, 181A Huntingdon Road, Cambridge, CB3 0DJ UK 3Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1055 USA 4Dipartimento di Biologia di Base ed Applicata, Università di L'Aquila, 67100 L'Aquila, Italy 5Istituto G.Donegani, 28100 Novara, Italy 6CNR, Centro di Studi sulla Biologia Molecolare, c/o Dipartimento di Scienze Biochimiche, Università di Roma ‘La Sapienza’, Piazzale A.Moro, 5, 00185 Roma, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:205-215https://doi.org/10.1093/emboj/cdg031 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 Å) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O2/H2O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme–substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure. Introduction Natural products such as the polyketide compounds tetracycline, erythromycin, daunorubicin and lovastatin produced by actinomycete bacteria and fungi have enormous therapeutic and commercial significance. Consequently, considerable research effort has been directed into study of the biosynthetic pathways that lead to these compounds in order to influence the nature and biological activity of the products produced (Hutchinson, 1997). This work has been aided by the cloning, sequence analysis and manipulation of the gene clusters responsible for the biosynthesis of aromatic and macrolide polyketides. Of particular interest have been enzymes that catalyse the assembly and subsequent cyclization of the polyketide chain (Hopwood and Sherman, 1990; Hutchinson and Fujii, 1995; Hopwood, 1997; Hutchinson, 1997). This has allowed many of the events surrounding carbon skeleton formation by condensation of simple building blocks such as a malonyl-CoA or methylmalonyl-CoA to be uncovered, but has left the identity and function of the enzymes that perform the later steps on these pathways relatively unclear. Late-acting, ‘tailoring’ enzymes catalyse a wide range of modifications to polyketide structure, such as hydroxylations, methylations or glycosylations. It is often these subtle changes to the overall molecular shape or charge that give the polyketide molecule its biological activity. A better understanding of the mechanistic and structural parameters of these enzymes may enable engineered biosynthesis (Townsend, 1997) to develop molecules with novel or altered biological activity or a better toxicological profile. The intense blue colour of the actinorhodin complex produced by Streptomyces coelicolor first attracted researchers into close study of this organism and into developing many of the genetic tools required for genetic manipulation of actinomycete bacteria. These genetic studies have reached their pinnacle with the completion of the entire 8.67 Mb genome sequence of this organism (Bentley et al., 2002). The gene cluster responsible for the biosynthesis of actinorhodin has long been thought of as the paradigm type II polyketide biosynthetic cluster (Malpartida and Hopwood, 1984, 1986). Analysis of the products of genetic mutations and sequence similarities to genes in other biosynthetic pathways allowed enzyme activities encoded by the gene cluster to be assigned tentatively (Hallam et al., 1988; Caballero et al., 1991, 1992; Fernández-Moreno et al., 1991, 1992, 1994). How ever, difficulties in purifying these enzymes, coupled with inadequate knowledge of the pathways and lack of substrates, mean that few of these assignments have been confirmed with biochemical studies of the proteins. While the numbers of natural product gene clusters identified has increased significantly each year, relatively few protein biochemical studies are published. There are still fewer structural studies involving enzymes from polyketide biosynthetic pathways, with the effort focused mainly on the polyketide synthase enzymes or the analogous proteins from fatty acid biosynthesis: the crystal structure of malonyl-CoA acyltransferase (Serre et al., 1995), the solution structure of the actinorhodin ACP (Crump et al., 1997), the crystal structure of plant chalcone synthases (Ferrer et al., 1999; Jez et al., 2000) and the crystal structure of the 6-deoxyerythronolide synthase thioesterase domain (Tsai et al., 2001). The erythromycin cytochrome P450 hydroxylase EryF (Cupp-Vickery and Poulos, 1995) appears to be the only polyketide ‘tailoring’ enzyme structurally characterized to date. Our work has focused on the enzymes catalysing oxygenations of polyketide biosynthetic pathways, particularly those catalysing the oxidations that introduce hydroxy- or quinone functionality to type II aromatic polyketides. ActVA-Orf6 was characterized as a monooxygenase (Kendrew et al., 1997, 2000) from the actinorhodin pathway that catalyses the oxidation of phenolic compounds to the corresponding quinone. The reaction catalysed in vivo by ActVA-Orf6 (Figure 1) is the conversion of 6-deoxydihydrokalafungin (6-DDHK) to dihydrokalafungin (DHK), intermediates in the biosynthesis of actinorhodin. Here we describe the next stage of our investigation of this enzyme: the high-resolution crystallographic structure at 1.3 Å of the ActVA-Orf6 monooxygenase of S.coelicolor A3 (2) in its native form and in complex with substrate and product analogues. This enzyme is one of a number of homologous monooxygenases (including, for example, TcmH, Shen and Hutchinson, 1993; and ElmH, Rafanan et al., 2001) from Streptomyces species catalysing the oxygenation of their various substrates without the need for prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. This is the first X-ray crystal structure of an enzyme from the actinorhodin biosynthetic pathway and in this perspective is particularly interesting since it allows us to propose a molecular mechanism for an unusual enzymatic reaction and to begin to develop ideas as to how the various tailoring enzymes from these biosynthetic pathways recognize their substrates. This allows us to develop a structural genomic approach to the functional and structural prediction of homologous proteins from other biosynthetic pathways. Figure 1.The oxidation of 6-deoxydihydrokalafungin (6-DDHK) to dihydrokalafungin (DHK) catalysed by ActVA-Orf6 monooxygenase. Download figure Download PowerPoint Results The overall structure ActVA-Orf6 monooxygenase crystallizes as a homodimer, as previously suggested (Kendrew et al., 2000) and consistent with previous biochemical studies (Kendrew et al., 1997). The monomers in the asymmetric unit are stabilized by an extensive hydrophobic interface with a buried surface of 3300 Å2. Each monomer belongs to the α–β sandwich group of the α + β fold class and comprises a ferredoxin-like split βαβ-fold (Orengo and Thornton, 1993), with two βαβ motifs forming an antiparallel sheet in which strand B1 lays in between strands B3 and B4 (Figure 2A). A search of the Protein Data Bank carried out with DALI (Holm and Sander, 1993) yields members of the ferredoxin-like fold family and indicates that sequence identity is lower than 15% over ≤90 structurally equivalent amino acids, pointing towards evolutionary convergence, which is not unusual for this type of fold. Figure 2.Structural model of ActVA-Orf6 monooxygenase. (A) The B subunit of ActVA-Orf6 monooxygenase viewed from the active site entrance is characterized by a ferredoxin fold, with four β-strands (B1–B4) and four α-helices (A1–A4). The blue strand is the C-terminal domain swap contributed by the A subunit. The mobile loop 34–38 (black arrow), the type II β-turn (red arrow) and the type III β-turn (blue arrow) are highlighted. Residues involved in the enzymatic mechanism are drawn as ball-and-stick. (B) The monomer–monomer interface. The interaction between the two β-sheets (yellow and cyan) is stabilized through a network of inter- and intramolecular hydrogen bonds, which also restrain the N-terminus (residues 2 and 4). Thick and thin sticks represent residues contributed, respectively, by the two subunits. (C) The homodimer of ActVA-Orf6 with nanaomycin D bound to subunit A, in the same orientation as in (F). (D) The homodimer of ActVA-Orf6. The 2-fold axis of symmetry is perpendicular to the plane of the picture (cross). (E) The homodimer of ActVA-Orf6 with nanaomycin D bound to subunit A, in the same orientation as in (G). (F) The bottom of the ActVA-Orf6 active site in the B subunit (yellow van der Waals spheres) shows a narrow tunnel (arrow) gated by Gln37 (purple). Ile110 (orange), belonging to the C-terminal swap of the other monomer, also contributes to the closure of the back of the active site. C- (red) and N- (blue) termini are also shown. (G) The dimer in a different orientation, showing nanaomycin D bound to subunit A (dark grey van der Waals spheres) of ActVA-Orf6 monooxygenase and exposing part of the pyrano-γ-lactone ring to the solvent. This moiety (and C-15 methyl, in particular) obstructs the active site entrance after binding. Download figure Download PowerPoint The surface of each monomer of ActVA-Orf6 monooxygenase is made up by polar amino acids exposed to the solvent, with a clustering of positive side chains (Arg73, Arg86 and Lys100) around the active site crevice, and negatively charged ones (Glu3, Glu30, Glu38, Glu42, Glu67 and Asp6) on the opposite side of the protein. This is consistent with the requirement for attracting a negatively-charged substrate towards the active site of the enzyme. A β-turn (Figure 2A, residues: Val43, Pro44, Gly45 and Phe46) positioned between helix A2 and strand B2 can be ascribed to the class of type II β-turns (Lewis et al., 1973). This is not a general feature of ferredoxin-like proteins, which can adopt different conformations for the β-turn in this topological position. Pro44 and Gly45 are strictly conserved among all the homologues of ActVA-Orf6 (Figure 3) and have the highest propensity for these positions in type II β-turns (Hutchinson and Thornton, 1994). Figure 3.Multiple sequence alignment of proteins similar to ActVA-Orf6 monooxygenase. The sequences have been selected from the output of a PSI-BLAST search performed using ActVA-Orf6 monooxygenase as query. Not all hits are shown in the alignment, and the alignments are less reliable from residue 75. Above the alignment, the secondary structure of ActVA-Orf6 is shown. The genes are clustered in three groups: a family of monooxygenases with an interface identical to ActVA-Orf6 monooxygenase (orange frame); a second family of putative Streptomyces monooxygenases that probably possess a different interface (green frame); and hypothetical proteins of unassigned function (blue frame). Aligned sequences: protein, organism (gene name and accession No. in SWALL database in parentheses): coelico, ActVA-Orf6 monooxygenase, S.coelicolor (ActVA-Orf6, Q53908); glauces, TcmF1 monooxygenase, S.glaucescens; (TcmH, P39889); olivace, TcmF1 monooxygenase, S.olivaceus (ElmH, Q9L4Y0); violace, C-5 anthrone oxidase, S.violaceus (Jad-Orf7, Q56157); str. C5, oxygenase, unspeciated streptomycete strain C5 (Streptomyces sp. C5; from the Frederick Cancer Research Council) (DauA-OrfE, Q55222); galilae, aklanonic acid anthrone monooxygenase, S.galilaeus (AknX, Q9L552); nogalat, SnoB protein, S.nogalater (SnoB, Q54493); peuceti, anthraquinol monooxygenase, S.peucetius (Dps-Orf8, Q54813); HY coeN, N-terminus of hypothetical 26.4 kDa protein, S.coelicolor (SCI7.27C, Q9X9W3); HY coeC, C-terminus of the same 26.4 kDa protein, S.coelicolor; HY loti, hypothetical protein, Mesorhizobium loti (MLR8211, Q983R7); HY aeru, hypothetical protein, Psuedomonas aeruginosa (PA2274, Q9I1K0). The biochemical function has been assigned experimentally for ActVA-Orf6 monooxygenase from S.coelicolor, TcmH from S.glaucescens, ElmH from S.olivaceus and AknX from S.galilaeus. For all the other proteins, the function has been deduced on the basis of homology with characterized proteins, although other indirect evidence may be available. Full boxes highlight conserved residues: purple, residues essential for tertiary structure (β-turns); red, interface polar residues (quaternary structure); yellow, active site residues; cyan, other identities. Similarities are only reported if they are consistent with structural data: light purple (tertiary structure) and light red (interface). End of the alignment: the first column reports percentage identity calculated for the ‘ferredoxin unit’ lacking helix A4 and the N-terminus (ID%/65, residues 10–74 of ActVA-Orf6 monooxygenase); the second column refers to the ‘minimal structural/functional unit’ (ID%/37, residues 38–74). Download figure Download PowerPoint A β-turn between strand B3 and helix A3 (Figure 2A, residues: Ser68, Glu69, Gln70 and Ala71) can be defined as a type III β-turn (Crawford et al., 1973; Lewis et al., 1973). The classical hydrogen bond interaction between the carbonyl of the first residue of the turn and the amide proton of the fourth residue is absent in this case. On the contrary, the side chain γ-oxygen and the carbonyl of Ser68 are hydrogen bonded to the amide group of Ala71 and Tyr72, respectively. These two interactions seem to stabilize the backbone of N-terminal residues of helix A3. The multiple alignment (Figure 3) shows that this serine is conserved throughout the homologues of ActVA-Orf6 or, alternatively, can be substituted by an aspartate residue, which is also a hydrogen bond acceptor and thus might play a similar role. The interface between the two monomers of ActVA-Orf6 is stabilized by hydrophobic interactions contributed by the β-sheets, with additional specific intermolecular contributions provided by Tyr63 and His52 of the two monomers, through tyrosine–tyrosine aromatic stacking and tyrosine–histidine hydrogen bonding (Figure 2B). Interestingly, these two residues are conserved throughout the homologous Tcm F1 monooxygenases from S.glaucescens (Shen and Hutchinson, 1993) and S.olivaceus (Rafanan et al., 2001), anthrone oxidase from S.venezuelae (Yang et al., 1996), and three hypothetical proteins of unassigned function identified in the complete genome sequences of S.coelicolor, Pseudomonas aeruginosa and Mesorhizobium loti (Figure 3). His52 has been implicated previously in catalysis and protein folding by site-directed mutagenesis studies (Kendrew et al., 1997). It now becomes clear that the vastly reduced activity displayed by the refolded mutant was due to disruption of the dimer interface rather than removal of vital catalytic residues. The sequence alignment shows that residues Ser48, Thr50 and Gln65 are also well conserved and appear to form a stabilizing intramolecular net of hydrogen bonds surrounding the interface residue Tyr63, also involving two water molecules (Figure 2B). The difference in gap distribution, degree of homology and conservation of interface residues across the alignment may indicate that there are two families of monooxygenases, with only one stabilized by a dimer interface that is similar to that displayed by ActVA-Orf6 monooxygenase. An attractive and clearly visible feature of the dimeric form of ActVA-Orf6 monooxygenase is a swapped strand whereby the C-terminal β-strand (B4, residues 103–113) contributes to the β-sheet of the other monomer by laying antiparallel to strand B2 (Figure 2A). This swap is essential for dimer stability, since its contribution to the buried surface of the interface is ∼2200 Å2 over a total of 3300 Å2. Importantly, the swapped strand takes part in the formation of the bottom of the active site, to which it contributes with Ile110 (Figure 2F). This segment is present in all the homologous sequences that conserve the residues involved in the dimerization contacts (Figure 3), apart from that of C-5 anthrone oxidase from S.violaceus, which is incomplete (Yang et al., 1996) at the C-terminus. Two different ferredoxin-like proteins, the bovine papillomavirus-1 E2 DNA-binding domain (Hegde et al., 1992) and muconolactone isomerase from Pseudomonas putida (Katti et al., 1989), are also dimeric; however, neither of the two displays the β-strand swapping observed in ActVA-Orf6 monooxygenase, and in the E2 DNA-binding domain the 2-fold symmetry axis is orthogonal to that observed in our case. In summary, examination of the three-dimensional structure suggests that ActVA-Orf6 monooxygenase is a new example of the widely represented ferredoxin-like fold, with a novel dimeric assembly. Furthermore, it is likely that the model will be a template for the tertiary structure of many homologous proteins across a range of microorganisms, and possibly for the quaternary structure of those proteins sharing the same polar residues involved in the dimer interface stabilization. The active site ActVA-Orf6 monooxygenase is a small enzyme (113 amino acid residues) that oxidizes a relatively large three-ringed aromatic substrate. Therefore, it is not surprising that all the secondary structure elements are involved in the formation of the active site. This is achieved by disrupting the centre of the helices of the two βαβ elements, resulting in the formation of two arches over a rigid floor contributed by the β-sheets (Figure 2A). The back of the active site cavity is closed by the fifth strand of the sheet and is provided by the other monomer. Within the active site, the accessible surface buried upon binding is ∼200 Å2 and applies to both the protein and the ligand, indicating that there is good shape complementarity. The high resolution and quality of the electron density maps confirm that ActVA-Orf6 monooxygenase, and presumably also the related proteins, does not require a metal ion or a prosthetic group for catalysis, as indicated by the biochemical data. In fact, there is no density that could be attributed to a metal ion, and no cluster of residues with a geometry that would provide a plausible metal coordination site. The native structure of ActVA-Orf6 monooxygenase (Figure 2A) suggests that four residues may be important for binding and/or catalysis: Tyr51, Asn62 and Trp66 belonging to the β-sheets, and Tyr72 hanging from α-helix A3. The latter residue clearly displays a double conformation on the A subunit, indicating a potential mobility that may be significant in the process of substrate binding or catalysis. Asn62 and Trp66 are conserved across the monooxygenase family, whereas Tyr51 and Tyr72 are less well conserved, perhaps reflecting the differences in the substrates utilized by the homologous enzymes in the family. In our crystals, the active site of subunit A is less accessible to solvent due to crystallographic packing and appears to be occupied by a polyethylene glycol (PEG) molecule; as we failed to obtain single crystals in the absence of PEG 200, this may be a requirement for crystal growth. To avoid introducing a model bias arising from incorrect positioning of this very mobile molecule, a PEG chain was not fitted to the electron density in the high-resolution model of the A subunit. However, good quality crystals containing a ligand in the A site were obtained by co-crystallization with nanaomycin D, a structural analogue of the natural product, and also with ANSA (1-anilino-8-naphthalenesulfonic acid; data not shown). This was found to bind within the active site of subunit A despite a molecular structure that is significantly different from that of the natural substrate. The most notable difference between the two monomers is the higher mobility of the region comprising amino acids 34–38 in subunit B (Figure 2A) with respect to subunit A. This difference in mobility is probably induced by crystal packing, but it may also play a functional role or at least points to an intrinsic mobility of the segment since it indicates that it can adopt two different conformations. In particular, in subunit A, Gln37 points towards the centre of the active site, making a hydrogen bond with Tyr51, a residue also implicated in binding or catalysis, whereas in subunit B it adopts a different conformation allowing a water molecule to enter and replace Gln37 in its interaction with the hydroxyl of Tyr51. In the latter conformation, movement of Gln37 opens a channel between the solvent and the bottom of the active site (Figure 2F). In the absence of the natural substrate, further understanding of the enzymic mechanism was achieved through the binding of substrate and product analogues to the active site. Four structures of ActVA-Orf6 monooxygenase have been solved (Table I), bound to sancycline and acetyldithranol (analogues of the substrate) or to nanaomycin D and the oxidized form of acetyldithranol (analogues of the product) (Figure 4). Nanaomycin D was co-crystallized with ActVA-Orf6 monooxygenase, whereas the other three analogues were diffused into native crystals. The two models of ActVA-Orf6 monooxygenase bound with reduced and oxidized acetyldithranol were obtained using the same preparation of soaked crystals, but freezing after 4 h and 2 weeks, respectively. This protocol was used because preliminary results showed that ActVA-Orf6 monooxygenase appeared (at least to some extent) to catalyse the oxidation of acetyldithranol (data not shown). This fits with other data indicating a broad substrate tolerance for these proteins (Kendrew et al., 1997). In fact, in crystals frozen after 2 weeks, the oxidized form of acetyldithranol can be seen bound at the active site (Figure 5A). Figure 4.Structure of the four ligands bound to ActVA-Orf6 crystals. Download figure Download PowerPoint Figure 5.Active site analysis. (A) 2Fo − Fc electron density map contoured at 1.0σ cut-off around oxidized acetyl dithranol and active site residues in the B subunit. (B) 2Fo − Fc electron density map contoured at 1.0σ cut-off around nanaomycin D in the A site. (C and D) Superposition in two different orientations of active sites from the five models of ActVA-Orf6 and significant hydrogen bond distances. Pink, native active site B; dark red, nanaomycin D in active site A (A = 2.9, B = 2.6, C = 2.4, D = 2.4, E = 2.8, F >13 Å); cyan, sancycline bound to the B site (A = 3.1, B = 3.1, F = 4.1 Å); green, acetyl dithranol bound to the B site (A = 3.3, B = 3.4, F = 3.5 Å); yellow, oxidized acetyl dithranol in the B site (A = 3.1, B = 3.4, F = 2.7 Å). The water molecule is only seen when nanaomycin D is bound to active site A. The different conformations of Gln37 in the two sites are clearly visible. The side chain oxygen of Gln37 is hydrogen-bonded to Tyr51 in the A site (dark red) and replaced by a water molecule (not shown) in the B site of all the other structures. Arg86 is forced out of the active site in the A subunit (dark red) by crystallographic packing. In the B subunit, it moves towards Tyr72 after diffusion of the ligands (green and yellow). A complete model of the dimer including nanaomycin D and side chains as in (C) and (D) is presented in the Supplementary data available at The EMBO Journal Online. Download figure Download PowerPoint Table 1. Summary of crystallographic and refinement statistics of ActVA-Orf6 monooxygenase Native Sanca AcetDit AcDOX Nana D K2PtCl4 Mers Mers 1 Data collection Space group: P212121 Res. limit (Å) 1.30 1.74 1.70 1.90 2.24 2.30 2.80 2.20 Total refl. (n) 798 091 118 991 228 029 64 564 68 040 Unique refl.b (n) 50 368 21 456 22 894 16 795 10 268 Completeness (%) 99.8 99.9 100.0 99.6 99.9 100.0 99.8 100.0 (last shell) 100.0 98.6 99.8 99.4 99.0 Rmergec 0.059 0.071 0.025 0.052 0.127 0.087 0.069 0.058 (last shell) 0.136 0.329 0.098 0.235 0.591 <I>/<σ>d 36.6 21.0 99.6 19.4 12.4 (last shell) 12.2 3.8 18.3 2.9 2.1 Soak. time (days) 5 0.5 14 45 7.5 21 MIRAS analysis No. of sites 4 2 1 Rcullise 0.87 0.95 0.88 Rcullis anoe 0.95 0.93 0.80 Phasing powerf 0.90 0.62 0.70 Low res. (Å) 20.0 15.0 20.0 20.0 20.0 High res. (Å) 1.30 1.74 1.70 1.90 2.24 Solvent atoms 258 179 158 121 60 Work reflections 47 745 20 327 21 673 15 686 9739 Free reflections 2560 1098 1170 834 493 Rcrystg 0.142 0.190 0.206 0.202 0.214 Rfreeg 0.168 0.240 0.246 0.256 0.272 R.m.s.d. bonds (Å) 0.013 0.015 0.015 0.015 0.013 R.m.s.d. angles (°) 1.99 2.50 2.66 2.50 2.80 a Derivatives of ActVA-Orf6: Sanc, sancycline; AcetDit, acetyl dithranol; AcDOX, oxidized acetyl dithranol; Nana D, nanaomycin D; Mers, Mers 1, mersalyl. b Unique reflections with I/σ>1, where I is the measured intensity and σ is the error of the intensity. c Rmerge = Σhkl Σ|Ij(hkl) − <I(hkl)>|/ΣhklΣj<I(hkl)>, with Ij(hkl) representing the intensity of measurement j and <I(hkl)> the mean of measurements for the reflection hkl. d <I >/<σ> = ratio between the mean intensity and the mean error of the intensity. e Rcullis =