Dependent Enzyme Moaa and Its Implications for Molybdenum Cofactor Deficiency in Humans

Home , Biotin synthase

Crystal structure of the S-adenosylmethionine- dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans

Petra Ha¨ nzelmann and Hermann Schindelin*

Department of Biochemistry and Center for Structural Biology, State University of New York, Stony Brook, NY 11794-5115

Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved July 21, 2004 (received for review June 28, 2004) The MoaA and MoaC proteins catalyze the first step during molyb- neously occupied by N and O atoms from the methionine moiety denum cofactor biosynthesis, the conversion of a guanosine de- of the cofactor. rivative to precursor Z. MoaA belongs to the S-adenosylmethionine MoaA shares 14% and 11% identity in the N-terminal region (SAM)-dependent radical enzyme superfamily, members of which with BioB and HemN, respectively, but is completely unrelated catalyze the formation of protein and͞or substrate radicals by with these proteins in the C-terminal region, which is in MoaA reductive cleavage of SAM by a [4Fe–4S] cluster. A defined in vitro characterized by another Cys-rich signature motif. Recently, it system is described, which generates precursor Z and led to the could be shown that human MOCS1A in fact assembles two -identification of 5؅-GTP as the substrate. The structures of MoaA in oxygen-sensitive [4Fe–4S] clusters, one typical for SAM the apo-state (2.8 Å) and in complex with SAM (2.2 Å) provide dependent radical enzymes and an additional one unique to valuable insights into its mechanism and help to define the defects MoaA proteins (4). The structure of MoaC has been determined caused by mutations in the human ortholog of MoaA that lead to earlier, and the protein was found to be present as a hexamer molybdenum cofactor deficiency, a usually fatal disease accompa- composed of three dimers with a putative active site located at nied by severe neurological symptoms. The central core of each the dimer interface (12). Because some of the SAM-dependent subunit of the MoaA dimer is an incomplete triosephosphate radical enzymes require another protein onto which the radical isomerase barrel formed by the N-terminal part of the protein, is transferred, it has been speculated that MoaC might act in a which contains the [4Fe–4S] cluster typical for SAM-dependent similar function. Here, we describe the structure of MoaA, the radical enzymes. SAM is the fourth ligand to the cluster and binds final player in the Moco biosynthetic pathway (13). In addition, to its unique Fe as an N͞O chelate. The lateral opening of the an in vitro system for precursor Z synthesis is reported, which led incomplete triosephosphate isomerase barrel is covered by the to the identification of 5Ј-GTP as the substrate. C-terminal part of the protein containing an additional [4Fe–4S] cluster, which is unique to MoaA proteins. Both FeS clusters are Methods separated by Ϸ17 Å, with a large active site pocket between. The Cloning, Expression, and Purification. See Supporting Text, which is noncysteinyl-ligated unique Fe site of the C-terminal [4Fe–4S] published as supporting information on the PNAS web site. cluster is proposed to be involved in the binding and activation of 5؅-GTP. Crystallization of MoaA. Crystals were grown under anaerobic conditions inside a glove box (Coy Laboratory Products, Ann Ͻ n humans, genetic deficiencies of enzymes involved in molyb- Arbor, MI) containing 2 ppm O2 with the hanging drop vapor diffusion technique by incubating MoaA (34 mg͞ml, 100 mM Idenum cofactor (Moco) biosynthesis lead to the pleiotropic ⅐ ͞ loss of the molybdoenzymes sulfite oxidase, aldehyde oxidase, Tris HCl, pH 9.0 300 mM NaCl) with 10 mM DTT for 30 min and xanthine dehydrogenase and trigger an autosomal recessive on ice and subsequent mixing in a 1:1 ratio with precipitant solution [90 mM Na Hepes, pH 7.5͞3.15 M Na formate͞3% and usually fatal disease, which is characterized by severe ͞ neurological symptoms (1, 2). The first step in Moco biosynthe- (vol vol) DMSO]. Crystals appeared within 2 days and were cryoprotected by soaking in mother liquor containing 30% sis, the conversion of a guanosine derivative to precursor Z, an ͞ (vol vol) glycerol. They belong to space group P212121 with cell oxygen-sensitive 6-alkyl pterin with a cyclic phosphate, is cata- ϭ ϭ ϭ lyzed by MoaA and MoaC (MOCS1A and MOCS1B in humans) dimensions of a 48.1, b 102.4, and c 191.2 Å and contain (3, 4). As in the pathways of folate, riboflavin, and biopterin two molecules per asymmetric unit (57% solvent content). synthesis, a guanosine derivative serves as the initial biosynthetic precursor (5, 6), but in contrast to all other pathways, its C8 atom Data Collection and Structure Determination. Fe–multiwavelength is retained and incorporated in a rearrangement reaction as the anomalous diffraction data were collected on a single crystal on first carbon of the precursor Z side chain. a Rigaku (Tokyo) RU-H3R rotating-anode x-ray generator MoaA belongs to the family of S-adenosylmethionine (SAM)- equipped with double focusing mirror optics and an R axis II dependent radical enzymes, members of which catalyze the imaging plate detector at a wavelength of 1.5418 Å and on formation of protein and͞or substrate radicals by reductive beamline X26C at the National Synchrotron Light Source at cleavage of SAM by a [4Fe–4S] cluster (7–9). Members of this Brookhaven National Laboratory (Upton, NY) on a Quantum family are involved in various metabolic processes, but until now 4R ADSC charge-coupled device detector at a wavelength of only two members have been structurally characterized: Biotin

synthase (BioB) (10), which converts dethiobiotin to biotin and This paper was submitted directly (Track II) to the PNAS ofﬁce. coproporphyrinogen III oxidase (HemN) (11), which catalyzes Abbreviations: SAM, S-adenosylmethionine; 5Ј-dA, 5Ј-deoxyadenosine; TIM, triosephos- the conversion of coproporphyrinogen III to protoporphyrino- phate isomerase; BioB, biotin synthase; HemN, coproporphyrinogen III oxidase; Moco, gen IX during heme biosynthesis. These enzymes display related molybdenum cofactor; MOCS, molybdenum cofactor synthesis. folds in their N-terminal regions, which also harbor the oxygen- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, labile [4Fe–4S] cluster. This cluster is ligated by only three Cys www.pdb.org (PDB ID codes 1TV7 and 1TV8). residues in the apoenzyme, whereas in the complex with SAM, *To whom correspondence should be addressed. E-mail: [email protected]. the vacant coordination site on one of the Fe atoms is simulta- © 2004 by The National Academy of Sciences of the USA

12870–12875 ͉ PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404624101 Downloaded by guest on October 1, 2021 1.7406 Å (Fe peak). All data sets were indexed, integrated, and scaled with HKL software (14). For subsequent calculations, the CCP4 suite was used (15) with exceptions as indicated. Four Fe sites were located with SOLVE (16), which was also used for phase refinement. Phases were subsequently improved with RESOLVE (17), and a model was built with the program O (18). The second monomer was generated with the NCS operation. The structure was refined with REFMAC5 incorporating translation, libration, and screw-rotation displacement (TLS) refinement in all cycles (19, 20). Each monomer was refined separately, and solvent molecules were automatically added with ARP (21). The MoaA–SAM complex structure was obtained by soaking crystals cocrystallized with 5 mM SAM in precipitant solution containing 10 mM SAM and 30% (vol͞vol) glycerol for 30 min. Data were collected on beamline X26C at the National Syn- chrotron Light Source, Brookhaven National Laboratory at a wavelength of 1.1 Å. The structure was solved by difference Fourier techniques and refined as described for apo-MoaA. Information concerning data collection and refinement sta- tistics is available in Tables 1 and 2, which are published as supporting information on the PNAS web site. Figures were created with MOLSCRIPT (22), BOBSCRIPT (23), PYMOL (DeLano Scientific, San Carlos, CA), SPOCK (24), and RASTER3D (25).

Assays of Precursor Z Synthesizing Activity and Reductive Cleavage of SAM. Nitrate reductase overlay assays and in vivo assays in moaA (KB2037) and moaC (KB2066) mutant cells were conducted as Fig. 1. In vitro synthesis of precursor Z. (A Left) Synthesis of precursor Z determined by measurement of its stable oxidized derivative, compound Z, by described (3). In vitro assays were performed under anaerobic BIOCHEMISTRY reversed-phase HPLC. MoaA (50 ␮M) and MoaC (50 ␮M) were incubated under conditions at room temperature in a total volume of 180 ␮lof100 ⅐ ͞ anaerobic conditions in the presence of 2 mM DTT with a 10-fold molar excess mM Tris HCl, pH 9.0 300 mM NaCl in the presence of 2 mM Ј Ј of SAM, 5 -GTP, MgCl2, and Na2S2O4 for 45 min (standard assay). (Top to DTT and a 10-fold molar excess of SAM, 5 -GTP, MgCl2, bottom) Reconstituted MoaA; anaerobically puriﬁed MoaA; without MoaC. ␮ Na2S2O4, and 50 M MoaC. The reaction was started by adding (Right) Time dependence of precursor Z formation (F) and reductive cleavage 50 ␮M MoaA and terminated, at specified times, by the addition of SAM in the presence of 5Ј-GTP generating 5Ј-dA (E). (B) Requirements for of 220 ␮l 100 mM Tris⅐HCl, pH 7.2͞50 ␮l of acidic iodine to precursor Z synthesis. The maximal amount of compound Z formed in a convert precursor Z to its stable fluorescent product compound standard assay (control, see A) was set to 100%. 5Ј-GTP was absent from the Z. After incubation at room temperature for 14 h, compound Z experiment with 5Ј-GDP and 5Ј-GMP. was purified as described (3). SAM cleavage was performed in a total volume of 160 ␮l in the presence of 2 mM DTT and a Ј obically purified MoaA with Ϸ4 mol of Fe per mol of protein 10-fold molar excess of SAM, 5 -GTP, and Na2S2O4. The reaction was started by adding 50 ␮M MoaA and terminated by shows activities of only 10–12% identifying MoaA with the two the addition of 40 ␮l of 50% (wt͞vol) trichloroacetic acid. SAM reconstituted [4Fe–4S] clusters as the catalytically competent and 5Ј-deoxyadenosine (5Ј-dA) were analyzed by reversed-phase form. Precursor Z synthesis is significantly increased in the ͞ presence of 2 mM DTT (Fig. 1B), although the specific function HPLC in 50 mM NH4H2PO4, pH 2.5, containing 10% (vol vol) methanol. of DTT is unknown. Precursor Z synthesis depends strictly on the presence of MoaA and MoaC. The reduction of the FeS Results and Discussion cluster(s) with Na2S2O4 is necessary to induce the cleavage of Ј Ј • Functional Characterization of MoaA and MoaC. MoaA and MoaC SAM yielding a 5 -dA radical (5 -dA ), as is the case in other Staphylococcus aureus SAM-dependent radical enzymes (8, 9). The reductive cleavage from were heterologously expressed as Ϸ His-tagged proteins in Escherichia coli. MoaA was purified of SAM is increased by a factor of 10 in the presence of Ј Ј under anaerobic conditions and additionally treated with Fe 5 -GTP. A time dependence of 5 -dA generation and precursor and L-cysteine in the presence of the cysteine desulfurase IscS. Z formation shows a simultaneous increase with a total yield of Ј ͞ This resulted in a protein with Ϸ9 mol of Fe per mol protein, 0.65 mol of 5 -dA mol of MoaA (Fig. 1A). indicating the assembly of two extremely oxygen-sensitive [4Fe–4S] clusters in analogy with MOCS1A (4). In solution, Structure of the MoaA Monomer. MoaA was crystallized under MoaA exists as a monomer (41 kDa) and dimer (82 kDa), as anaerobic conditions, and the structure was solved by multi- determined by size exclusion chromatography (data not wavelength anomalous diffraction techniques using Fe as the shown). MoaC is mainly in a hexameric form (120 kDa), which, anomalous scatterer. The final model, refined at 2.8-Å resolution ϭ ϭ however, easily dissociates into the dimer (40 kDa). For both (Rcryst 18.9%, Rfree 21.1%) contains 327 (Gln-3 to Gln-329) proteins, the functional oligomeric states are unknown. In vitro of 340 residues. The 3 N-terminal residues and the His-tag as well assembly studies in the presence and absence of substrates and as 11 C-terminal residues are disordered. The central core of crosslinking experiments, as well as coexpression and subse- each monomer, which is formed by the N-terminal part of the quent copurification, failed to identify a tight MoaA–MoaC protein (amino acids 3–223), is composed of an incomplete ␣␤ protein complex. [( )6] triosephosphate isomerase (TIM) barrel (27) with a ␣␤ Purified MoaA and MoaC are able to catalyze the formation lateral opening (Fig. 2A). A canonical TIM barrel [( )8] of precursor Z in a 5Ј-GTP- and SAM-dependent reaction (Fig. consists of an inner ring of eight parallel ␤-strands that is 1); however, neither 5Ј-GDP nor 5Ј-GMP can be used. Precursor wrapped by an outer wheel comprised typically of eight ␣-helices. Z was identified by coelution with precursor Z isolated from E. However, some proteins display deviations from the classical ␣␤ coli chlM cells known to accumulate this compound (26). Anaer- ( )8-barrel fold, including variations in the number of

Ha¨ nzelmann and Schindelin PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 ͉ 12871 Downloaded by guest on October 1, 2021 unique to MoaA poteins but similar to the N-terminal cluster, it does not feature a Cys ligand to the fourth Fe atom.

Structure and Function of the MoaA Dimer. MoaA is present as an elongated homodimer with dimensions of 79 Å ϫ 58 Å ϫ 45 Å (Fig. 2 B and C). The two monomers are related by a noncrys- tallographic 2-fold symmetry axis, which is approximately parallel to the axis of the barrel. The contact interface covers an area of Ϸ1,400 Å2, corresponding to Ϸ9% of the surface area of each monomer. The dimer interface is mainly formed by contacts between helices ␣7 of the N-terminal domain and ␣10 of the C-terminal domain of each monomer and is composed of polar (41%) as well as hydrophobic (59%) residues. As described above, the enzyme exists in both a monomeric and dimeric form in solution, but the functional significance of the dimer is not entirely clear at present, because residues located in the dimer interface region are not conserved in MoaA proteins. The C-terminal ␣-helix (␣10) is located in the dimer interface and points toward the active site of the second monomer (Fig. 2 B and C). The conformation of this helix is indicative of helix swapping, a known mechanism in oligomeric assembly (29). MoaAs from eubacteria and eukaryotes contain a functionally important double Gly motif at the C terminus (3). Deletion of both Gly residues affects binding and redox properties of the FeS cluster(s), suggesting a location in the active site (Fig. 7A, which is published as supporting information on the PNAS web site). However, there is no interpretable electron density for the last 11 amino acids, indicating that these residues are highly mobile. It is possible that the C terminus adopts an ordered conformation only upon binding of 5Ј-GTP. Based on the crystal structure, there are two possible conformations of the C terminus: either it points back into the active site of the same monomer, or, more likely, into the active site of the other monomer. The C terminus is in both cases at a distance of Ϸ20–23 Å to the noncysteinyl ligated Fe site of the C-terminal FeS cluster. Maybe the last C-terminal amino acids protrude into the hydrophilic channel Fig. 2. Overall structure of MoaA. (A) Ribbon diagram of the monomer. The Ј N-terminal TIM barrel structure is shown in maroon, and the C-terminal part leading to closure of the active site after 5 -GTP-binding (Fig. is shown in gold. (B) Ribbon diagram of the dimer. The two monomers are 2C). In contrast to eubacteria and eukaryotes, precursor Z colored in blue and orange. (C) Molecular surface of the dimer colored as in B synthesis in archaea does not depend on a C-terminal double Gly viewed along the hydrophilic channel in one of the monomers. FeS clusters are motif and, interestingly, at least MoaAs from Methanococcus shown in CPK (Fe, green; S, yellow), and SAM is shown in stick representation. jannaschii and Archaeoglobus fulgidus are exclusively in a monomeric form (unpublished data). These data indicate differ- ences in the oligomeric state and catalytic mechanism of ar- ␤ -strands, their orientation (typically all parallel), local struc- chaeal MoaAs. tural distortions, and also lack of barrel closure (28). The C-terminal part of the protein (amino acids 224–329) covers the The Active Site of MoaA and Substrate Binding. The 2.2-Å resolution lateral opening of the barrel (Fig. 2A). This part consists of a (R ϭ 21.3, R ϭ 24.1) structure of MoaA in complex with ␤ cryst free three-stranded antiparallel -sheet, an extended loop leading to SAM revealed no conformational changes, because the apo and the other side of the protein and three ␣-helices, which complete SAM complex structures can be superimposed with an rms the C-terminal part. Interestingly, a hydrophilic channel is deviation of 0.35 Å. The bound SAM constitutes the fourth observed in the center of the TIM barrel. ligand to the [4Fe–4S] cluster located in the N-terminal domain MoaA contains two [4Fe–4S] clusters, one in the N-terminal and binds as an N͞O chelate to the unique Fe position of this part and one in the C-terminal part of the protein (Fig. 2A). Both cluster through its amino-group nitrogen (N-Fe 2.6 Å) and FeS clusters are on opposite sides of the hydrophilic channel. carboxyl-group oxygen (O-Fe 1.9 Å) (Fig. 3A). In addition, the The N-terminal cluster is ligated by three Cys residues, which sulfonium sulfur of SAM is at a distance of 3.3 Å to the same Fe originate from a signature sequence motif (Cx3Cx2C) character- and 3.6 Å to the adjacent sulfur atom. The SAM-binding site istic of all SAM-dependent radical enzymes (8, 9). This motif is comprises residues from disparate parts of the sequence and 3D contained in a 31-residue loop extending from ␤-strand 1 to structure (Figs. 3A and 6). However, amino acid residues sur- ␣-helix 1 of the TIM barrel (Fig. 6, which is published as rounding the bound SAM within a distance of 5 Å are conserved supporting information on the PNAS web site), and the 3 Cys in MoaA proteins. SAM is bound in extended conformation coordinate 3 of the 4 Fe of the [4Fe–4S], whereas the fourth Fe stretching across the top of the barrel and is stabilized by has a vacant protein coordination site in the apoenzyme. In the comparatively few interactions, including four hydrogen bonds. structure of apo-MoaA, a yet-unidentified residual difference The net result of these interactions is that SAM is completely density is present at the noncysteinyl ligated Fe site that is too buried from solvent and appears ideally positioned for electron strong to be modeled as a single water molecule. The second transfer from the [4Fe–4S] cluster. This binding mode is anal- [4Fe–4S] cluster is ligated by a Cx2Cx13C motif and is assembled ogous to what has been observed in BioB and HemN (10, 11). between two loops in the C-terminal part of the protein leading Residual difference density close to the FeS–SAM substrate to a closure of the barrel structure. This [4Fe–4S] cluster is complex, which is also present in the apo-structure, could be

12872 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404624101 Ha¨ nzelmann and Schindelin Downloaded by guest on October 1, 2021 BIOCHEMISTRY

Fig. 3. The active site of MoaA. (A) Stereoview of the FeS clusters and SAM. SAM is shown in a FoϪFc omit map contoured at 3 ␴. Hydrogen bonds are indicated as dashed lines. (B) Closer view of the active-site pocket created by predominantly positively charged amino acids. Conserved amino acids are labeled in red. Secondary structural elements and cofactors are rendered transparent. (C) Surface representation viewed from both sides into the active site channel. Conserved residues are mapped onto the molecular surface. (D) ␴A weighted 2FoϪFc map of the C-terminal FeS cluster with a DTT molecule as the fourth ligand contoured at 1 ␴. Surrounding amino acids are shown as a C␣ trace colored in orange. Amino acids, FeS clusters, and SAM are shown in ball and stick.

modeled as a phosphate or sulfate (not shown). This anion is at An interesting feature of the MoaA structure is a residual distance of Ϸ5 Å to the Fe coordinating the SAM and the 3Ј-OH difference density associated with the unique Fe site of the group of SAM. Because no phosphate͞sulfate-containing buff- C-terminal FeS cluster, which could be modeled as DTT (Fig. ers were used during purification, reconstitution, and crystalli- 3D). DTT binds with one of its sulfur atoms to the fourth Fe zation, this anion must be already bound tightly before isolation atom (S4–Fe4 2.2 Å), thus also resulting in a tetrahedral of the protein. Accordingly, it is stabilized by several hydrogen coordination of this Fe atom. The remainder of the DTT is not bonds with D128 (O4-N 2.8 Å, O1-O␦1 3.0 Å, and O3-O␦2 2.9 involved in any contacts with the protein. As described above, Å), and Y30 (O3-OH 3.2 Å). So far, no functional role is known synthesis of precursor Z is significantly increased in the presence for the involvement of this anion. of DTT. Maybe, DTT helps in vitro to stabilize the labile The two FeS clusters define the general location of the active nonligated Fe and maintains it in the ferrous state. During site of MoaA. The closest distance between them is Ϸ17 Å catalysis DTT is presumably replaced by the substrate (see (Fe4–Fe4), and they create a large active site pocket interrupted below). Furthermore, it has been shown in case of a ferredoxin by the hydrophilic channel. The active site is constructed from that binding of DTT to the [4Fe–4S] cluster led to a 200 mV predominantly basic residues (Fig. 3 B and C), including five negative shift of the redox potential of the FeS cluster (32). highly conserved Arg and Lys side chains, which are ideally In addition to their primary function in mediating electron positioned to compensate the negative charges of the 5Ј-GTP redox processes, FeS clusters are known to control protein phosphate groups. Attempts to get a structure with bound structure, act as environmental sensors, serve as modulators of 5Ј-GTP either by cocrystallization or soaking experiments have gene regulation, and also participate in radical generation (33), failed so far. Presumably, the 5Ј-GTP interaction is weak as as is the case for the FeS cluster common to all SAM-dependent already observed for the second substrate SAM. Additionally, a radical enzymes. A structural role or function in electron transfer decreased binding can be expected due to the high salt concen- of the C-terminal FeS cluster is considered unlikely as the cluster trations necessary for crystal growth. is easily degraded and has an incomplete cysteinyl ligation. A Although the substrate for precursor Z synthesis could be putative function of this MoaA specific FeS cluster could be identified as 5Ј-GTP, the MoaA structure revealed no typical analogous to the N-terminal FeS cluster that is involved in nucleotide binding motif͞fold like a P-loop (30) or Rossmann anchoring the substrate SAM to position it in a proper confor- fold (31) frequently found in dinucleotide binding proteins. This mation for the subsequent radical chemistry. One can imagine is not unexpected because during precursor Z synthesis the that the unique Fe site of the C-terminal FeS cluster anchors the pyrophosphate moiety of 5Ј-GTP is only transiently attached to 5Ј-GTP to position the guanine C8 atom close to the 5Ј-C atom the protein and subsequently released. Binding and release of of the 5Ј-dA radical (5Ј-dA•) generated by the N-terminal FeS pyrophosphate usually requires a divalent cation like Mg2ϩ or cluster. The spatial proximity could propagate the radical based Mn2ϩ as a cofactor. Somewhat surprisingly, precursor Z synthe- reaction by abstracting a hydrogen atom from the C8 atom sis does not depend on MgCl2 or MnCl2 (Fig. 1B), and there is leading to the proposed rearrangement reaction (5, 6). This no obvious cation-binding site. function in substrate binding would be analogous to aconitase,

Ha¨ nzelmann and Schindelin PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 ͉ 12873 Downloaded by guest on October 1, 2021 not resolved in the structure. To obtain insights into the molecular mechanisms leading to human Moco deficiency, site- directed mutagenesis was used to generate the described missense mutations in the gene encoding MOCS1A. The activity of these variants was analyzed with a nitrate reductase overlay assay. None of the resulting variants were able to complement an E. coli moaA mutant (Fig. 7B). All identified missense mutations led to a loss of activity of MOCS1A, and the purified proteins show a decrease in Fe content by Ϸ20–50% and different UV-visible absorption spectra, suggesting changes in the envi- ronment of the FeS clusters (Fig. 7C). The MoaA structure revealed that all affected residues are located in the active-site pocket (Fig. 4). G273 (G324 in MOCS1A) has main chain dihedral angles characteristic of left-handed helices (␾ ϭ 72.5°, ␺ ϭ 43.7°), which are uncommon for non-Gly residues. G273 is located in a turn connecting two Fig. 4. Location of missense mutations detected in Moco-deﬁcient patients. ␤ Secondary structure elements and cofactors are rendered transparent. Human -strands that lead into the two loops ligating the C-terminal FeS mutations, FeS clusters, and SAM are shown as ball and stick. cluster. Disruption of this turn by a G324E mutation presumably results in major conformational changes and subsequently in loss of the C-terminal FeS cluster leading to MOCS1A destabiliza- where it could be shown that an FeS cluster participates in the tion and enhanced proteolytic degradation. These structural enzymatic mechanism (34, 35). Aconitase assembles like MoaA considerations are in agreement with the fact that this mutant a [4Fe–4S] cluster ligated by only three Cys and the noncysteinyl- protein cannot be purified in a stable form (Fig. 7C), and that the ligated unique Fe site is involved in the binding and activation loss of the C-terminal FeS cluster destabilizes the protein (4). of substrates. Mutation of the three active site residues, R73͞R17, R123͞R71, and R319͞R268, presumably affects the proposed interaction Location of Missense Mutations Identified in Moco-Deficient Patients. with the negatively charged phosphate groups of 5Ј-GTP. G75͞ Mutations within the MOCS1A ORF that is homologous to G127 is hydrogen bonded to the SAM amino nitrogen (Fig. 3A) MoaA account for Ϸ50% of all known cases of Moco deficiency and ensures its correct orientation. The mutations G126D and (1, 2). All of the genetic lesions affect highly conserved regions G127R in this loop region presumably result in loss of SAM–FeS (Fig. 6). Several mutations are found in close proximity to the binding. The specific roles of these disease-associated amino conserved Cys motifs of the N- and C-terminal [4Fe–4S] clusters acids in the catalytic mechanism remain to be investigated. (R73W, R123W, R319Q, and G324E; S. aureus R17, R71, R268, and G273). The R319Q mutation is the most common mutation, Structural Comparison of Members of SAM-Dependent Radical En- accounting for 14% of Moco-deficient alleles and 21% of all zymes. A search for structural homologs of MoaA with the identified mutations. Furthermore, missense mutations were program DALI (36) identified, besides a large number of TIM found in the conserved 123RxTGGEP129 sequence (S. aureus barrel proteins, the two SAM-dependent radical enzymes BioB 71RxTGGEP77) and additionally in the catalytically essential and HemN as the closest structural homologs. HemN was the C-terminal double Gly motif (G384S; S. aureus G339) (3) that is highest match with a Z score of 11.7 and an rms deviation of 4.7

Fig. 5. Structural comparison of SAM-dependent radical enzymes and reactions catalyzed. ␣-Helices are colored in orange, ␤-sheets are colored in blue, and turns are colored in gray. Nonstructurally conserved secondary structural elements are rendered transparent. FeS clusters and substrates are shown in ball and stick.

12874 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404624101 Ha¨ nzelmann and Schindelin Downloaded by guest on October 1, 2021 Å for 232 residues, whereas BioB was the second highest match to which subfamily MOCS1A͞MoaA belongs. Because two with a Z score of 11.3 and an rms deviation of 4.1 Å for 208 proteins, MoaA͞MOCS1A and MoaC͞MOCS1B, are essential residues. The structures of BioB (10), HemN (11), and MoaA for precursor Z synthesis, one could speculate that MoaA͞ show in their N-terminal parts a common structural core in- MOCS1A is the activating enzyme that leads to the generation volved in [4Fe–4S] cluster and SAM binding (Fig. 5). Never- of catalytically active glycyl- and thiyl-radicals on MoaC͞ theless, in each case, unique structural elements near the C MOCS1B. Based on the structure of E. coli MoaC, a putative terminus and additional cofactors͞substrates are required for active site has been mapped that is located at the dimer interface the generation of radicals on structurally divergent substrates. (12). Four sequence stretches, including the highly conserved ␣␤ Common to all structures is a complete [( )8, BioB] or incom- amino acids G117, C141, and G175 in MOCS1B, are involved. ␣␤ plete [( )6, MoaA, HemN] TIM barrel ligating the [4Fe–4S]– Site-directed mutagenesis of these amino acids led only in the SAM substrate complex. BioB assembles an additional air stable case of G175 to a complete inactivation (Fig. 8, which is [2Fe–2S] cluster, the postulated source of sulfur for biotin published as supporting information on the PNAS web site). formation. The nonhomologous C-terminal part of BioB with Additionally, the G117A variant does not show the typical two additional ␣-helices and ␤-strands completes the TIM accumulation of precursor Z, although wild-type levels of molyb- barrel. HemN contains a second SAM molecule immediately dopterin are present. Therefore, G175 and G117 are functionally ͞ adjacent to the first, and both SAM molecules were proposed to and or structurally essential and could be involved in harboring be involved in catalyzing the two propionate decarboxylation a catalytic and essential glycyl radical. However, it is unlikely that ͞ reactions. Besides this, a distinct C-terminal domain that is MoaC MOCS1B belongs to subfamily I, because (i) generation missing in the BioB and MoaA structures covers the lateral of a G175A variant in the homologous E. coli MoaC resulted only ͞ opening of the incomplete TIM barrel. Finally, MoaA ligates a in an activity decrease by 40% (12), (ii) MoaC MOCS1B does ͞ second oxygen-sensitive [4Fe–4S] cluster by only three Cys not contain the typical radical sequence motif [consensus RV(C residues, with the unique Fe site proposed to be involved in S)GY] (9), (iii) MOCS1B has no catalytically essential Cys Ј residue, and (iv) MoaC (12) does not show the conserved 3D anchoring the substrate 5 -GTP. A search for structural ho- ␣͞␤ mologs of the C-terminal part of MoaA with the program DALI structure with a central 10-stranded barrel as known from (36) revealed no structurally significant homolog. members of this family (37). Although precursor Z synthesis depends strictly on MoaC͞MOCS1B, there is so far no obvious ͞ Investigations of a Putative Function of Human MOCS1B in a Radical- function for MoaC MOCS1B. Based on these considerations, it

is unlikely that during the formation of precursor Z, a protein- BIOCHEMISTRY Based Reaction Mechanism. SAM-dependent radical enzymes can ͞ be divided into two subfamilies (8, 9). Subfamily I is a two- based radical mechanism is involved and MoaA MoaC can protein system composed of an FeS protein, which is the therefore be classified as the first member of a third subfamily activating protein catalyzing the reductive cleavage of SAM into (subfamily III), characterized by an accessory protein that is not methionine and the 5Ј-dA radical (5Ј-dA•), which then generates involved in the radical-based mechanism. a glycyl radical on the catalytic protein. The glycyl radical in turn creates a thiyl radical. Representative members of this subfamily This work was supported by National Institutes of Health Grant DK54835 (to H.S.). The National Synchrotron Light Source in are anaerobic ribonucleotide reductase and pyruvate formate Brookhaven is supported by the Department of Energy and the National lyase. BioB and HemN belong to subfamily II, which is a Institutes of Health, and beamline X26C is supported in part by the single-protein system combining activating and catalytic activi- Research Foundation of the State University of New York at Stony ties by direct substrate radical formation. So far, it is not known Brook.

1. Reiss, J. (2000) Hum. Genet. 106, 157–163. 19. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997) Acta Crystallogr. D 53, 2. Reiss, J. & Johnson, J. L. (2003) Hum. Mutat. 21, 569–576. 240–255. 3. Ha¨nzelmann, P., Schwarz, G. & Mendel, R. R. (2002) J. Biol. Chem. 277, 20. Winn, M. D., Isupov, M. N. & Murshudov, G. N. (2001) Acta Crystallogr. D 57, 18303–18312. 122–133. 4. Ha¨nzelmann, P., Herna´ndez, H. L., Menzel, C., Garcı´a-Serres, R., Huynh, 21. Perrakis, A., Morris, R. & Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458–463. B. H., Johnson, M. K., Mendel, R. R. & Schindelin, H. (2004) J. Biol. Chem. 22. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946–950. 279, 34721–34732. 23. Esnouf, R. M. (1997) J. Mol. Graphics 15, 132–134. 5. Wuebbens, M. M. & Rajagopalan, K. V. (1995) J. Biol. Chem. 270, 1082–1087. 24. Christopher, J. A. & Baldwin, T. O. (1998) J. Mol. Graphics 16, 285. 6. Rieder, C., Eisenreich, W., O’Brien, J., Richter, G., Gotze, E., Boyle, P., 25. Merritt, E. A. & Bacon, D. J. (1997) Methods Enzymol. 277, 505–524. Blanchard, S., Bacher, A. & Simon, H. (1998) Eur. J. Biochem. 255, 24–36. 26. Johnson, M. E. & Rajagopalan, K. V. (1987) J. Bacteriol. 169, 117–125. 7. Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. 27. Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia, C. (1995) J. Mol. Biol. (2001) Nucleic. Acids Res. 29, 1097–1106. 247, 536–540. 8. Jarrett, J. T. (2003) Curr. Opin. Chem. Biol. 7, 174–182. 28. Nagano, N., Orengo, C. A. & Thornton, J. M. (2002) J. Mol. Biol. 321, 741–765. 9. Frey, P. A. & Magnusson, O. T. (2003) Chem. Rev. 103, 2129–2148. 29. Liu, Y. & Eisenberg, D. (2002) Protein Sci. 11, 1285–1299. 10. Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T. & Drennan, C. L. (2004) 30. Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990) Trends Biochem. Sci. 15, Science 303, 76–79. 430–434. 11. Layer, G., Moser, J., Heinz, D. W., Jahn, D. & Schubert, W. D. (2003) EMBO J. 22, 6214–6224. 31. Rossmann, M. G., Moras, D. & Olsen, K. W. (1974) Nature 250, 194–199. 12. Wuebbens, M. M., Liu, M. T., Rajagopalan, K. V. & Schindelin, H. (2000) 32. Butt, J. E., Sucheta, A., Armstrong, F. A., Breton, J., Thomson, A. J. & Structure Fold. Des. 8, 709–718. Hatchikiant, E. C. (1993) J. Am. Chem. Soc. 115, 1413–1421. 13. Rizzi, M. & Schindelin, H. (2002) Curr. Opin. Struct. Biol. 12, 709–720. 33. Johnson, M. K. (1998) Curr. Opin. Chem. Biol. 2, 173–181. 14. Otwinowski, Z. & Minor, W. (1997) Methods Enzymol. 276, 307–326. 34. Kent, T. A., Dreyer, J.-C., Kennedy, M. C., Huynh, B. H., Emptage, M. H., 15. Collaborative Computational Project 4 (1994) Acta Crystallogr. D 50, 760–763. Beinert, H. & Mu¨nck, E. (1982) Proc. Natl. Acad. Sci. USA 79, 1096–1100. 16. Terwilliger, T. C. & Berendzen, J. (1999) Acta Crystallogr. D 55, 849–861. 35. Beinert, H., Kennedy, M. C. & Stout, C. D. (1996) Chem. Rev. 96, 2335–2373. 17. Terwilliger, T. C. (2000) Acta Crystallogr. D 56, 965–972. 36. Holm, L. & Sander, C. (1995) Trends Biochem. Sci. 20, 478–480. 18. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991) Acta Crystallogr. 37. Eklund, H. & Fontecave, M. (1999) Structure (Cambridge, U.K.) 7, R257– A 47, 110–119. 262.

Ha¨ nzelmann and Schindelin PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 ͉ 12875 Downloaded by guest on October 1, 2021