From the
Department of Plant Biology, Technical
University Braunschweig, 38023 Braunschweig, Germany and the
¶Department of Biochemistry, Duke University
Medical Center, Durham, North Carolina 27710
Received for publication, March 26, 2003 , and in revised form, May 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor (2). 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 (5, 6). 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 (7). In the last step of Moco biosynthesis in humans, molybdenum is incorporated into MPT by the two-domain protein gephyrin (8, 9). 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 (10). Recently a third group has been identified with genetic defects in gephyrin (11).
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 (12, 13). 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 (12). The high resolution crystal structure of E. coli MPT synthase has been solved and showed a heterotetrameric structure for the enzyme (14, 15). 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 (14, 15). 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 (16, 17). Subsequently, the MoaD thiocarboxylate group is formed by the action of a NifS-like protein using L-cysteine as the ultimate sulfur source (18). Human MOCS3 exhibits homologies to E. coli MoeB and is presumed to activate MOCS2A in a similar manner.
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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 (19). 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.
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EXPERIMENTAL PROCEDURES |
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Cloning, Expression, and Purification of MOCS2A and MOCS2B
The genes encoding MOCS2A and MOCS2B were amplified by PCR from a human cDNA
library (8). 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 A150 in MOCS2B were introduced using the
Transformer kit from Clontech. To create the
143 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 A600 = 0.5
with 300 mM isopropyl--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--D-thiogalactopyranoside to 0.1 mM when
the cultures had attained A600 = 0.6. Following 56
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 (22). 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 AssayFor genetic complementation
of E. coli moaD and moaE mutants, plasmids pSL173, pSL203,
pSL204, and pSL206 and the mutated versions pSL173-E168K, pSL173-A150,
and pSL173-
143 were transformed into these strains. Nitrate
reductase activity was analyzed by a colony overlay assay
(23).
Size Exclusion ChromatographySize 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 0.21.0 ml and immediately injected into the column without preincubation.
MPT Synthase ReactionsPrecursor Z was purified from an E. coli moaD mutant and quantitated as previously described (24). 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 (25). 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 x 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 SpectroscopyCD 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.
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RESULTS |
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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. coliFor 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 (26) and adapted for the purification of carboxylated and thiocarboxylated E. coli MoaD by Gutzke et al. (12) and Wuebbens and Rajagopalan (13). 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.
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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 SubunitsIt 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 (12, 13). 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 (12). However, it is possible that MOCS2A has an anomalous elution behavior from the size exclusion column as was shown for E. coli MoaE (13), 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 ComplexesUsing 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).
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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).
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Generation of Mutations in MOCS2A and MOCS2B Based on Group B
PatientsTo 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
(2). It has been reported that
most group B patients are very severely affected
(2); however, one patient
identified with an unusually mild form of the disease was heterozygous for two
single-base substitutions in MOCS2A
(19). 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 (19). 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.
2 and
19 and
Fig. 1A), and amino
acid exchange E168K and a deletion of Ala150 were introduced into
MOCS2B (Ref. 27 and
Fig.
1B).2
Amino acid alignments of MOCS2B with homologous proteins from different
sources revealed that eukaryotic MOCS2B proteins include an N-terminal
extension of 40 amino acids not found in any of the eubacterial
homologues and without homology to any protein characterized in the data base
(7). To gain insights into the
role of the N terminus of MOCS2B, a MOCS2B variant without the first 43 amino
acids was also created.
To analyze the functionality of the MOCS2B variants, mutated versions of
MOCS2B in pET15b were transformed into the E. coli moaE
mutant and tested for functional complementation of this strain. Because
complementation of the E. coli moaD- strain with wild type
MOCS2A was not observed (Fig.
1D), it was not possible to analyze the MOCS2A variants
by this method. As shown in Fig.
5A, MOCS2B variants E168K and A150 did not
complement the E. coli moaE mutant. Only the
143 MOCS2B
variant was able to complement the E. coli moaE mutant, although at a
lower level compared with wild type MOCS2B
(Fig. 5A).
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For purification of MOCS2A and MOCS2B variants, the same expression and purification conditions were applied as described for the wild type proteins. Although purification of MOCS2A-V7F produced about 2 mg of carboxylated or 1 mg of thiocarboxylated protein/liter of culture (Fig. 5B), significant amounts of MOCS2A-S15R could not be obtained even after varying the expression conditions. Immunodetection of expressed MOCS2A-S15R with MOCS2A antisera indicated that the variant had been expressed as a fusion protein but at a reduced level compared with wild type MOCS2A (data not shown), so it is possible that this variant is unstable after cleavage with dithiothreitol or ammonium sulfide.
Purification of the MOCS2B variant E168K resulted in about 8 mg of purified
protein/liter of E. coli (Fig.
5B). In contrast, attempts to purify MOCS2B-A150
failed because the protein formed inclusion bodies upon expression in the
E. coli cells. The
143 variant of MOCS2B is also
unstable because the majority of the protein was rapidly degraded during or
after purification (Fig.
5B). Because MOCS2A-V7F and MOCS2B-E168K were the only
protein variants that were successfully expressed and purified in a stable
form, all further characterizations were performed with these two
proteins.
CD Spectroscopy of Purified MOCS2A-V7F and MOCS2B-E168K
VariantsTo analyze the influence of these two mutations on the
overall folding of their respective proteins, both the variant and wild type
subunits were subjected to CD spectroscopy. The CD spectrum recorded for
MOCS2A-V7F showed a slightly increased intensity between 185200 and
210230 nm compared with wild type MOCS2A, indicating differences in its
-helical and
-strand composition that point toward small
conformational differences (data not shown). In contrast, the CD spectra of
MOCS2B and MOCS2B-E168K were almost identical, showing that this mutation did
not alter the overall folding of the protein (data not shown).
Analysis of the Activity of MOCS2A-V7F and MOCS2B-E168K VariantsThe ability of the two variants to associate with their corresponding large and small subunits to form a heterotetrameric MPT synthase complex was examined. Size exclusion chromatography experiments showed that the MOCS2A-V7F variant did not form a complex with MOCS2B in either its carboxylated (Fig. 6A) or thiocarboxylated form (data not shown). In contrast, the MPT synthase tetramer was readily formed in mixtures of MOCS2B-E168K with equimolar amounts of MOCS2A as seen in Fig. 6B. To determine whether MPT synthases containing mutant subunits are able to convert precursor Z to MPT in vitro, the activity of mixtures containing the variants was compared with wild type MPT synthase. As seen in Fig. 6C, the activity of the MOCS2A-V7F/MOCS2B mixture was very low, because only 10% of maximal MPT was produced after an incubation time of 120 min. Interestingly, the formation of a recently described hemisulfurated MPT intermediate (13) was observed in this mixture as shown in the dotted trace of Fig. 6C. The MOCS2A/MOCS2B-E168K mixture produced about 50% of maximal MPT after an incubation time of 120 min (Fig. 6C). During the time course of the wild type MPT synthase reaction, the amount of form A in the samples decreased after incubation times greater than 60 min. This apparent decrease is explained by the instability of free MPT, which is rapidly degraded after release from MPT synthase. The affinity of MOCS2B-E168K and the MOCS2A/MOCS2B-E168K mixture for precursor Z was also determined. Only 10% of the precursor Z bound to MOCS2A/MOCS2B was found in the MOCS2A/MOCS2B-E168K mixture, and MOCS2B-E168K alone exhibited no precursor Z binding (data not shown). Thus, the decreased activity observed with this variant is likely to be the result of decreased substrate binding.
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DISCUSSION |
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In this report we describe the successful purification of human MOCS2A and MOCS2B after heterologous expression in E. coli. Like E. coli MoaD and MoaE, separately purified MOCS2A and MOCS2B readily assemble to form a heterotetrameric MPT synthase complex. Comparison of the activities of human MPT synthase to E. coli MPT synthase showed that the human synthase converts precursor Z to MPT at a much slower rate than the E. coli synthase. Studies with chimeric MPT synthases showed that human and E. coli small and large subunits are somewhat interchangeable. MPT synthase complexes containing MoaE are significantly faster than mixtures containing MOCS2B (Fig. 3), indicating that the turnover rate of MPT synthase is dependent on the source of the large subunit.
With the development of an in vitro assay for human MPT synthase,
it was of interest to evaluate the effects of mutations in MPT synthase small
and large subunits leading to Moco deficiency. Only a few group B patients
have been identified to date, and they are generally very severely affected by
the disease (2). However, one
patient identified with an unusually mild form of the disease, harbored a
valine to phenylalanine exchange at the N terminus of MOCS2A
(19), and it was speculated
that a low level of residual activity from the V7F allele might be responsible
for the milder clinical symptoms
(19). CD spectroscopy of the
purified MOCS2A-V7F variant showed that the mutation altered the protein
structure sufficiently to produce noticeable differences in both its
-strand and
-helical composition. The crystal structure of E.
coli MPT synthase (14,
15) has revealed that the
N-terminal portion of MoaD forms a
-strand that is quite distant from
the putative active site at its C-terminal glycine. The moaD gene
lacks 15 of the first 18 bases present in MOCS2A, and the MoaD
protein has an isoleucine at position 2 corresponding to Val7 in
MOCS2A. Introducing the bulkier phenylalanine at position 2 instead of valine
would disrupt the structure at the N terminus of the small subunit, indirectly
affecting enzyme activity
(19). Our analyses support and
extend this statement by documenting that the structure of MOCS2A-V7F is
altered in a manner that disturbs its interaction with MOCS2B and results in
accumulation of a one-sulfur intermediate
(13). This suggests that the
V7F mutation hinders transfer of the second sulfur to precursor Z. Despite
this, the residual rate of MPT formation that was observed here is presumably
sufficient to support enough sulfite oxidase activity to moderate the clinical
symptoms in this patient.
The second mutation analyzed in detail was found in a severely affected
patient heterozygous for a frameshift mutation, 7262, on one
MOCS2B allele and a point mutation affecting the first nucleotide of
MOCS2B exon 7 on the second allele that results in the E168K amino
acid exchange (27). Despite
the fact that Glu168 is one of the few extremely conserved amino
acids in MOCS2B (7), CD
spectroscopy indicated that the structure of this variant was not altered by
the amino acid exchange despite the substitution of a positive for a negative
charge. Moreover, the activity of this variant was higher than that for the
MOCS2A-V7F variant found in a less severely affected patient
(Fig. 6C). Our results
suggest that the E168K mutation attenuates the binding of precursor Z,
presumably by altering the binding pocket. This finding is consistent with the
location of this residue in E. coli MPT synthase (corresponding
number Glu128) where it is close to but not directly in the active
site and unlikely to be directly involved in the catalytic reaction
(14,
15). In a recent report
(13), E. coli MPT
synthase containing MoaE-E128K was shown to be 17-fold slower than the wild
type enzyme. Thus, the mutation affects activity in both human and E.
coli synthases, but the mutant protein from E. coli retains a
much smaller portion of its normal activity. Given the clinical outcome of
this mutation, the relatively high level of in vitro activity
observed with the human E168K protein points to a mutation-related RNA
processing defect as the causative factor in the severely affected patient
with this mutation. As suggested by Reiss et al.
(27), the G
A mutation
underlying the E168K substitution occurs at the first nucleotide of exon 7 in
MOCS2B and as such may have severe consequences on splicing at this
junction and on the overall expression of MOCS2B.
Two other mutations identified in two affected patients with severe
symptoms of Moco deficiency were generated in MOCS2A and MOCS2B. However,
neither the MOCS2A-S15R variant nor the MOCS2B-A150 variant could be
purified after heterologous expression in E. coli. It remains
speculative whether these amino acid exchanges also influence protein
stability in humans, thus leading to Moco deficiency. Amino acid alignments of
MOCS2B with homologous proteins from different sources revealed that
eukaryotic MOCS2B proteins harbor an N-terminal extension of
40 amino
acids not found in any of the eubacterial homologues
(7). It was speculated that the
N-terminal extension in MOCS2B might serve as a cell localization signal that
is cleaved off after protein targeting. If this were the case, then the
processed protein should retain its activity when expressed in E.
coli. However, because our results showed that the
MOCS2B-
143 variant is not expressed in an isolable form, it
appeared that the N terminus of MOCS2B may be required for protein stability
in E. coli. Immunodetection of endogenous MOCS2B in human tissues
could be used to determine whether MOCS2B is processed in humans.
In the future, the established expression system for human MOCS2A and MOCS2B in E. coli will be used for the generation of further site-specific mutants identified in Moco-deficient group B patients. Comprehensive biochemical, enzymatic, and molecular analysis of these proteins will help to define the specific underlying defects caused by mutations in human MPT synthase that lead to Moco deficiency. Crystallization of human MPT synthase should also shed light on the functions of specific amino acid residues and help explain differences in the reaction mechanism of human and E. coli MPT synthase.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 49-531-391-5885; Fax:
49-531-391-8208; E-mail:
S.Leimkuehler{at}tu-bs.de.
1 The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin;
HPLC, high performance liquid chromatography; MOCS2A-SH, thiocarboxylated
MOCS2A; MOCS2A-OH, carboxylated MOCS2A.
2 J. L. Johnson, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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