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

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Dependent Enzyme Moaa and Its Implications for Molybdenum Cofactor Deficiency in Humans 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 September 29, 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.
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