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Mechanistic Studies of Human Molybdopterin Synthase Reaction and Characterization of Mutants Identified in Group B Patients of Molybdenum Cofactor Deficiency

2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês

10.1074/jbc.m303092200

1083-351X

Silke Leimkühler, Andrea Freuer, José Angel Santamaria Araujo, K.V. Rajagopalan, Ralf R. Mendel,

Peptidase Inhibition and Analysis

Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z. Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z. Molybdenum cofactor (Moco) 1The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; HPLC, high performance liquid chromatography; MOCS2A-SH, thiocarboxylated MOCS2A; MOCS2A-OH, carboxylated MOCS2A. biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum (1Rajagopalan K.V. Johnson J.L. J. Biol. Chem. 1992; 267: 10199-10202Abstract Full Text PDF PubMed Google Scholar). Moco is essential for the activity of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase in humans (2Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (98) Google Scholar). Human Moco deficiency leads to the pleiotropic loss of all three of these molybdoenzymes and usually progresses to death at an early age (3Johnson J.L. Wadman S.K. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2271-2283Google Scholar). Isolated sulfite oxidase deficiency is a related disease in which molybdenum cofactor biosynthesis is normal, but sulfite oxidase activity is altered (4Johnson J.L. Duran M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3163-3177Google Scholar). The clinical symptoms of molybdenum cofactor deficiency are indistinguishable from those of isolated sulfite oxidase deficiency with the notable exception that xanthinuria is absent in the latter (4Johnson J.L. Duran M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3163-3177Google Scholar). In both cases, affected neonates show feeding difficulties, neurological abnormalities such as attenuated brain growth, untreatable seizures, dislocated ocular lenses in most cases, and death in early childhood. Although milder symptoms are occasionally observed, none of the treatments tested to date have produced consistent improvement of the clinical status (4Johnson J.L. Duran M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3163-3177Google Scholar). Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor (2Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (98) Google Scholar). The MOCS1 locus encodes two proteins homologous to Escherichia coli MoaA and MoaC that are needed for the formation of precursor Z, which were shown to have an unusual bicistronic structure with open reading frames for both MOCS1A and MOCS1B in a single transcript (5Gray T.A. Nicholls R.D. RNA. 2000; 6: 928-936Crossref PubMed Scopus (50) Google Scholar, 6Hänzelmann P. Schwarz G. Mendel R.R. J. Biol. Chem. 2002; 277: 18303-18312Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Similarly, the MOCS2 locus encodes the two subunits of MPT synthase and has been shown to be bicistronic with overlapping reading frames encoding MOCS2A and MOCS2B, the congeners of E. coli MoaD and MoaE (7Stallmeyer B. Drugeon G. Reiss J. Haenni A.L. Mendel R.R. Am. J. Hum. Genet. 1999; 64: 698-705Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the last step of Moco biosynthesis in humans, molybdenum is incorporated into MPT by the two-domain protein gephyrin (8Feng G. Tintrup H. Kirsch J. Nichol M.C. Kuhse J. Betz H. Sanes J.R. Science. 1998; 282: 1321-1324Crossref PubMed Scopus (349) Google Scholar, 9Stallmeyer B. Schwarz G. Schulze J. Nerlich A. Reiss J. Kirsch J. Mendel R.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1333-1338Crossref PubMed Scopus (139) Google Scholar). Based on the presence or absence of precursor Z in clinical samples, Moco deficiency was originally divided into two complementation groups. Group A patients have a defect at the MOCS1 locus and therefore do not produce precursor Z, whereas group B patients have a defect at the MOCS2 locus and are characterized by the accumulation of precursor Z (10Johnson J.L. Wuebbens M.M. Mandell R. Shih V.E. J. Clin. Invest. 1989; 83: 897-903Crossref PubMed Scopus (51) Google Scholar). Recently a third group has been identified with genetic defects in gephyrin (11Reiss J. Gross-Hardt S. Christensen E. Schmidt P. Mendel R.R. Schwarz G. Am. J. Hum. Genet. 2001; 68: 208-213Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because human MPT synthase shows significant homologies to E. coli MPT synthase (see Fig. 1, A and B), the mechanism of sulfur transfer to precursor Z is expected to be similar. The reaction mechanism of E. coli MPT synthase has been described in detail (12Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). E. coli MPT synthase is activated by formation of a thiocarboxylate group at the second glycine of its C-terminal Gly-Gly motif that serves as the direct sulfur donor for the formation of the dithiolene group in MPT (12Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The high resolution crystal structure of E. coli MPT synthase has been solved and showed a heterotetrameric structure for the enzyme (14Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar, 15Rudolph M.J. Wuebbens M.M. Turque O. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2003; 278: 14514-14522Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). MoaD, the small subunit of E. coli MPT synthase, shows a three-dimensional fold similar to ubiquitin and interacts via its C terminus with the large subunit, MoaE, thereby forming two hypothetical active sites in the heterotetramer (14Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar, 15Rudolph M.J. Wuebbens M.M. Turque O. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2003; 278: 14514-14522Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Prior to the formation of the MoaD C-terminal thiocarboxylate group, the protein is activated by adenylation of the C-terminal carboxylate, a reaction carried out by the E. coli MoeB protein (16Lake M.W. Temple C.A. Rajagopalan K.V. Schindelin H. J. Biol. Chem. 2000; 275: 40211-40217Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Leimkühler S. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 34695-34701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Subsequently, the MoaD thiocarboxylate group is formed by the action of a NifS-like protein using l-cysteine as the ultimate sulfur source (18Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Human MOCS3 exhibits homologies to E. coli MoeB and is presumed to activate MOCS2A in a similar manner. Here we describe the purification and characterization of human MPT synthase encoded by MOCS2A and MOCS2B. After separate purification, MOCS2A and MOCS2B were assembled in vitro to generate MPT synthase. The catalytic activity of this human MPT synthase was compared with in vitro assembled E. coli MPT synthase and with chimeric proteins assembled from mixtures of human and E. coli large or small subunits. A number of mutations in the MOCS2 proteins have been identified in group B patients. Whereas one patient described with an amino acid exchange in MOCS2A showed a particularly mild form of molybdenum cofactor deficiency, two patients with mutations identified in MOCS2B were severely affected (19Johnson J.L. Coyne K.E. Rajagopalan K.V. Van Hove J.L.K. Mackay M. Pitt J. Boneh A. Am. J. Med. Genet. 2001; 104: 169-173Crossref PubMed Scopus (33) Google Scholar). To analyze differences in symptoms of Moco deficiency based on varying MPT synthase activities, corresponding mutants in MOCS2A and MOCS2B have been generated and characterized. Bacterial Strains, Media, and Growth Conditions—The E. coli moaD(DE3) and moaE (RK5204) mutant strains used in this study were described previously (20Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 117-125Crossref PubMed Google Scholar, 21Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar). E. coli BL21(DE3) cells and pET15b were obtained from Novagen. E. coli strain ER2566(DE3), plasmid pTYB2, and chitin affinity resin were obtained from New England Biolabs. Cell strains containing expression plasmids were grown aerobically at either 30 °C (pET15b) or 16 °C (pTYB2) in LB medium in the presence of 150 μg/ml ampicillin or 50 μg/ml chloramphenicol. Sephadex 200 and Superose 12 matrix were purchased from Amersham Biosciences. Cloning, Expression, and Purification of MOCS2A and MOCS2B— The genes encoding MOCS2A and MOCS2B were amplified by PCR from a human cDNA library (8Feng G. Tintrup H. Kirsch J. Nichol M.C. Kuhse J. Betz H. Sanes J.R. Science. 1998; 282: 1321-1324Crossref PubMed Scopus (349) Google Scholar). For expression of MOCS2A, the gene was cloned into the NdeI and KpnI sites of pTYB2. For expression of MOCS2B, PCR primers were designed to allow cloning into the XbaI and BamHI sites of the multiple cloning site of pET15b, resulting in plasmid pSL173. The amino acid substitutions V7F and S15R in MOCS2A and E168K and A150Δ in MOCS2B were introduced using the Transformer kit from Clontech. To create the Δ1–43 N-terminal deletion of MOCS2B, a PCR primer was designed that exchanged serine 43 for a methionine, allowing direct cloning into the XbaI site of pET15b. For expression of all MOCS2A variants, the plasmids were transformed into E. coli ER2566(DE3) cells. The cells were grown at 37 °C in 6-liter cultures, and expression was induced at A 600 = 0.5 with 300 mm isopropyl-β-d-thiogalactopyranoside. Growth was continued for 18 h at 16 °C, and the cells were harvested by centrifugation. Cell lysis was achieved by several passages through a French pressure cell. After centrifugation, the supernatant was combined with 20 ml of chitin affinity resin equilibrated with 20 mm Tris-HCl, 0.5 m NaCl, 0.1 mm EDTA, 0.1% Triton X-100, pH 8.0, and stirred for 30 min at 4 °C. The resin was then poured into a column and washed with 100 ml of equilibration buffer without Triton X-100. For intein cleavage, the resin was incubated for 18 h with 20 ml of 250 mm Tris-HCl, 0.5 m NaCl, 0.1 mm EDTA, pH 8.5, containing 50 mm ammonium sulfide (MOCS2A-SH) or 50 mm dithiothreitol (MOCS2A-OH). Released MOCS2A was eluted with 30 ml of 250 mm Tris-HCl, 0.5 m NaCl, 0.1 mm EDTA, pH 8.5, and concentrated. Both proteins were exchanged into 100 mm Tris-HCl, pH 7.2, prior to use. For purification of MOCS2B, the protein was coexpressed with a plasmid containing the molecular chaperones GroES/EL. The groES/EL genes were first cloned into the NcoI and XhoI sites of pET15b before subcloning into the SphI and HindIII sites of pLysS (Novagen) to generate pPH67. Six-liter cultures of E. coli BL21(DE3) cells cotransformed with pSL173 and pPH67 were induced by the addition of isopropyl-β-d-thiogalactopyranoside to 0.1 mm when the cultures had attained A 600 = 0.6. Following 5–6 h of growth at 30 °C, the cultures were harvested by centrifugation and resuspended in 30 ml of 50 mm Tris-HCl, 1 mm EDTA, pH 7.5. Cell lysis was achieved by several passages through a French pressure cell. After centrifugation, the supernatant volume was increased to 150 ml with buffer prior to the addition of 16.5 ml of 20% (w/v) streptomycin sulfate. After centrifugation, MOCS2B was precipitated by the addition of 176 g/liter of ammonium sulfate. Precipitated protein was pelleted by centrifugation, resuspended in 50 mm Tris-HCl, 1 mm EDTA, pH 7.5, and dialyzed against the same buffer. Final purification of MOCS2B was achieved by chromatography on a Superose 12 gel filtration column equilibrated in 100 mm Tris-HCl, 200 mm NaCl, pH 7.2. All of the protein concentrations were determined using their calculated extinction coefficients at 280 nm. MOCS2A expression constructs for genetic complementation of the E. coli moaD mutant were cloned into pTrc-His (22Temple C.A. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). PCR primers were designed to allow cloning of MOCS2A or MocS2B into the NdeI and BamHI sites of the multiple cloning region of pTrc-His to generate pSL203 and pSL173, respectively. For coexpression of MOCS2A and MOCS2B, MOCS2B was PCR-amplified with primers that allowed cloning into the BamHI and HindIII sites of pSL203, resulting in pSL204. To facilitate coexpression of MOCS2A and MOCS2B, the E. coli Shine-Dalgarno sequence was attached to the 5′ end of MOCS2B. For coexpression of MOCS2A, MOCS2B, and MOCS3, MOCS3 was PCR-amplified with primers that allowed cloning into the HindIII site of pSL204, resulting in pSL206. To ensure coexpression of MOCS3 with MOCS2A and MOCS2B, the E. coli Shine-Dalgarno sequence was also attached to the 5′ end of MOCS3. The orientation of MOCS3 after cloning into pSL204 was checked by restriction analysis. Nitrate Reductase Overlay Assay—For genetic complementation of E. coli moaD and moaE mutants, plasmids pSL173, pSL203, pSL204, and pSL206 and the mutated versions pSL173-E168K, pSL173-A150Δ, and pSL173-Δ1–43 were transformed into these strains. Nitrate reductase activity was analyzed by a colony overlay assay (23Glaser J.H. DeMoss J.A. Mol. Gen. Genet. 1972; 116: 1-10Crossref PubMed Scopus (53) Google Scholar). Size Exclusion Chromatography—Size exclusion chromatography was performed at room temperature using a Sephadex 200 column (Amersham Biosciences) equilibrated in 100 mm Tris-HCl, 200 mm NaCl, pH 7.2. Small and large MPT synthase subunits were mixed in a volume of 0.2–1.0 ml and immediately injected into the column without preincubation. MPT Synthase Reactions—Precursor Z was purified from an E. coli moaD mutant and quantitated as previously described (24Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 1995; 270: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Routine MPT synthase reactions were performed at room temperature in a total volume of 400 μl of 100 mm Tris-HCl, pH 7.2. Small and large MPT synthase subunits were combined, and the reaction was started by precursor Z addition. At specified times, the reaction was terminated by the addition of 50 μl of acidic iodine to convert precursor Z to compound Z and MPT to form A (25Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar). Both products are stable fluorescent compounds that can be readily quantitated by HPLC analysis using a fluorescent detector. Following incubation at room temperature for 14 h, excess iodine was removed by the addition of 55 μl of 1% ascorbic acid, and the sample was adjusted with 1 m Tris to pH 8.3. The phosphate monoester of form A was cleaved by the addition of 40 mm MgCl2 and 1 unit of calf intestine alkaline phosphatase. Form A and compound Z obtained by this method were further purified on diethyl(2-hydroxypropyl)aminoethyl(diethyl(2-hydroxypropyl)aminoethyl) Sephadex A-25 columns with a 500 μl bed volume. Form A was eluted from these columns in a volume of 2 ml of 10 mm acetic acid, and compound Z was eluted in a volume of 5 ml of 100 mm HCl. The reactions were analyzed by subsequent injection (100 μl for form A and 200 μl for compound Z) onto a C-18 reversed phase HPLC column (4.6 × 250 mm; ODS-Hypersil; particle size, 5 μm) equilibrated with 50 mm ammonium acetate containing 10% methanol for form A or 10 mm potassium phosphate, pH 3.0, with 1% methanol for compound Z analysis (flow rate, 1 ml/min). In-line fluorescence was monitored by an Agilent 1100 series detector with excitation at 370 nm and emission at 450 nm. CD Spectroscopy—CD spectroscopy was performed with purified proteins in 40 mm potassium phosphate buffer, pH 7.6. The spectra were recorded on a J-800 CD-spectrometer (Jasco) at 20 nm/min scan speed with seven repetitions. Cloning of MOCS2A and MOCS2B and Test of Functional Complementation of E. coli moaD and moaE Mutants— MOCS2A and MOCS2B, the genes for human MPT synthase, are arranged in a bicistronic transcript with two overlapping reading frames (7Stallmeyer B. Drugeon G. Reiss J. Haenni A.L. Mendel R.R. Am. J. Hum. Genet. 1999; 64: 698-705Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In vitro translation and mutagenesis experiments demonstrated that MOCS2A and MOCS2B are translated independently, leading to the synthesis of the 9.8-kDa MOCS2A protein and the 20.8-kDa MOCS2B protein (7Stallmeyer B. Drugeon G. Reiss J. Haenni A.L. Mendel R.R. Am. J. Hum. Genet. 1999; 64: 698-705Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Although MOCS2A shows an identity of 27.5% to the E. coli MoaD protein (Fig. 1A), MOCS2B contains an N-terminal extension of ∼40 amino acids not found in any of the eubacterial homologues. The remainder of MOCS2B shows an identity of 29.3% to E. coli MoaE (Fig. 1B). For functional complementation studies of E. coli moaD and moaE mutants, MOCS2A and MOCS2B were cloned together or separately into E. coli expression vectors as shown in Fig. 1C. These were subsequently transformed into the mutant E. coli strains. Complementation of the strains by the human proteins would result in the production of active nitrate reductase, a Moco-containing enzyme, the activity of which is dependent on the ability of cells to synthesize Moco. Complementation can be scored on plates using an overlay assay for formate-dependent nitrate reductase activity (23Glaser J.H. DeMoss J.A. Mol. Gen. Genet. 1972; 116: 1-10Crossref PubMed Scopus (53) Google Scholar). As shown in Fig. 1D, although the E. coli moaE - cells were functionally complemented with MOCS2B, the E. coli moaD - cells were not complemented by either MOCS2A or by coexpression of MOCS2A with MOCS2B. To explore the possibility that MOCS2A is not activated by E. coli MoeB, a construct that coexpresses MOCS2A, MOCS2B, and MOCS3, the human equivalent of E. coli MoeB (Fig. 1C), was tested for its ability to complement the moaD - strain. As shown in Fig. 1D, coexpression of all three human proteins resulted in partial complementation of the E. coli moaD mutant, suggesting that MOCS2A cannot be activated by endogeneous E. coli MoeB. This failure could be due either to the inability of the two proteins to favorably interact or to slight differences between the mechanism of sulfur transfer in prokaryotic and eukaryotic cells. Expression and Purification of Separate MOCS2A and MOCS2B Subunits in E. coli—For purification of MOCS2A and MOCS2B, the proteins were cloned into separate expression vectors for heterologous expression in E. coli. To synthesize MOCS2A with either a carboxylate or a thiocarboxylate group at the C-terminal glycine residue, MOCS2A was cloned into the E. coli expression vector, pTYB2, resulting in a fusion protein containing both a C-terminal intein tag and a chitin-binding domain for affinity purification. Taking advantage of the intein-catalyzed self-cleavage reaction and the resulting transes-terification, cleavage of the product with dithiothreitol results in the carboxylated form of MOCS2A, whereas cleavage with ammonium sulfide results in the thiocarboxylated form of MOCS2A. This method was originally described for the purification of ThiS variants (26Kinsland C. Taylor S.V. Kelleher N.L. McLafferty F.W. Begley T.P. Protein Sci. 1998; 7: 1839-1842Crossref PubMed Scopus (42) Google Scholar) and adapted for the purification of carboxylated and thiocarboxylated E. coli MoaD by Gutzke et al. (12Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and Wuebbens and Rajagopalan (13Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). MOCS2A was purified from E. coli ER2566(DE3) cells resulting in 3 mg of carboxylated or 1.5 mg of thiocarboxylated protein/liter of culture. Both purified proteins exhibited an approximate monomeric mass of 10 kDa on Coomassie-stained SDS-polyacrylamide gels (Fig. 2A), which is in close correspondence to the calculated molecular mass of 9.8 kDa for MOCS2A. For purification of MOCS2B, the expression plasmid pSL173 was transformed into E. coli BL21(DE3) cells. To promote proper folding of MOCS2B, the cells were cotransformed with a plasmid expressing the E. coli heat shock chaperones GroES/EL under the control of the T7 promotor. MOCS2B was purified by ammonium sulfate precipitation and chromatography on a Superose 12 column as shown in Fig. 2B. This procedure yielded ∼10 mg of MOCS2B/liter of E. coli culture. The protein exhibited an apparent monomeric mass of 21 kDa on Coomassie-stained SDS gels that corresponded closely to the calculated molecular mass of 20.8 kDa for MOCS2B. Assembly of MOCS2A and MOCS2B from Single Subunits—It has been demonstrated that both the carboxylated and thiocarboxylated forms of MoaD readily associate with MoaE in vitro to form the heterotetrameric MPT synthase complex but that only the complex containing thiocarboxylated MoaD is active (12Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). To determine whether human MPT synthase can also readily be assembled from its separately purified subunits, MOCS2A and MOCS2B were mixed and analyzed for the generation of the MPT synthase tetramer by size exclusion chromatography. As shown in Fig. 2C, in the presence of excess MOCS2A, all of the MOCS2B was converted to the MPT synthase complex. The observed elution positions of the synthase complex and MOCS2B showed that MOCS2A and MOCS2B associate to form a heterotetrameric MPT synthase complex and that MOCS2B exists as a dimer in solution. The elution position of MOCS2A from the size exclusion column revealed that MOCS2A also forms a dimer in solution, unlike E. coli MoaD, which exists as a monomer in solution (12Gutzke G. Fischer B. Mendel R.R. Schwarz G. J. Biol. Chem. 2001; 276: 36268-36274Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, it is possible that MOCS2A has an anomalous elution behavior from the size exclusion column as was shown for E. coli MoaE (13Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), mimicking the behavior of a dimer. No differences were observed between the carboxylated or thiocarboxylated form of MOCS2A to assemble with MOCS2B. Analysis of the Activity of Assembled MPT Synthase Complexes—Using purified thiocarboxylated MOCS2A and MoaD, the ability of assembled human MPT synthase to convert purified precursor Z to MPT in vitro was compared with that of E. coli MPT synthase. In addition to homogeneous E. coli and human MPT synthase, chimeric MOCS2A/MoaE and MoaD/MOCS2B mixtures were also analyzed. MPT produced was quantitated by conversion to its fluorescent derivative form A. The time courses of the MPT synthase reactions are shown in Fig. 3. All of the MPT synthase combinations were able to form MPT from precursor Z in vitro but with varying efficiency. The reaction mixture containing E. coli MoaD/MoaE with precursor Z reached its end point after 30 s. The mixture of human MOCS2A/MOCS2B with precursor Z reached its maximum after 8 min of incubation. Bigger differences in reaction time were observed with chimeric MPT synthases (Fig. 3). Although the reaction of MOCS2A/MoaE MPT synthase reached its end point after 2 min, the mixture of MoaD/MOCS2B was significantly slower, and maximum MPT formation was reached after an incubation time of 60 min (data not shown). To see whether the differences in MPT synthase reaction rates could be explained by differences in affinity for the substrate, precursor Z binding to the proteins was examined. The MOCS2B and MoaE subunits in addition to mixtures of inactive MOCS2A/MOCS2B and MoaD/MoaE were incubated with excess precursor Z and subjected to gel filtration to remove unbound precursor Z from the mixture. Precursor Z bound to the excluded fraction was quantitated by conversion to its oxidized fluorescent derivative, compound Z. As shown in Fig. 4, although some precursor Z coeluted with MOCS2B (Fig. 4A), no precursor Z remained bound to MoaE after gel filtration (Fig. 4B). In contrast, identical amounts of precursor Z remained protein-bound in mixtures of inactive carboxylated MOCS2A or MoaD with MOCS2B and MoaE, respectively (Fig. 4). Generation of Mutations in MOCS2A and MOCS2B Based on Group B Patients—To date, about 100 patients have been diagnosed worldwide for Moco deficiency. Among these patients, the majority are in group A and only a few of the mutations identified in the patients have been located in the genes for either MOCS2A or MOCS2B (2Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (98) Google Scholar). It has been reported that most group B patients are very severely affected (2Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (98) Google Scholar); however, one patient identified with an unusually mild form of the disease was heterozygous for two single-base substitutions in MOCS2A (19Johnson J.L. Coyne K.E. Rajagopalan K.V. Van Hove J.L.K. Mackay M. Pitt J. Boneh A. Am. J. Med. Genet. 2001; 104: 169-173Crossref PubMed Scopus (33) Google Scholar). The mutation in one allele introduced a stop codon in place of a glutamine (Q6X), and the mutation in the second allele resulted in substitution of a valine for a phenylalanine (V7F). It was speculated that a low level of residual activity from the V7F allele might be responsible for the milder clinical symptoms of this patient (19Johnson J.L. Coyne K.E. Rajagopalan K.V. Van Hove J.L.K. Mackay M. Pitt J. Boneh A. Am. J. Med. Genet. 2001; 104: 169-173Crossref PubMed Scopus (33) Google Scholar). To analyze the molecular basis of base pair exchanges leading to molybdenum cofactor deficiency in group B patients, these mutations were introduced in either MOCS2A or MOCS2B by site-directed mutagenesis. Amino acid exchanges V7F and S15R were introduced into MOCS2A (Refs. 2Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (98) Google Scholar and 19Johnson J.L. Coyne K.E. Rajagopalan K.V. Van Hove J.L.K. Mackay M. Pitt J. Boneh A. Am. J. Med. Genet. 2001; 104: 169-173Crossref PubMed Scopus (33) Google Scholar and Fig. 1A), and amino acid ex