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INTRODUCTION |
Type 1 hereditary tyrosinemia
(HT1,1 OMIM 276700) is an
autosomal recessive disease caused by a deficiency of
fumarylacetoacetate hydrolase (FAH, EC 3.7.1.2), the last enzyme
involved in the tyrosine catabolic pathway. FAH catalyzes the
hydrolysis of fumarylacetoacetate into fumarate and acetoacetate (1).
FAH is mainly expressed in mammalian liver. It is also expressed, in
lesser amounts, in cells from a wide range of tissues such as kidneys,
adrenal glands, lungs, heart, bladder, intestine, stomach, pancreas,
lymphocytes (2), skeletal muscle, placenta, fibroblasts, chorionic
villi (3), and some glial cells of the mammalian brain (4). A deficiency of FAH causes the accumulation of succinylacetone (1), maleylacetoacetate, and fumarylacetoacetate (FAA), the latter presenting a mutagenic potential (5-7). Both an acute and a chronic form of the disease have been described on the basis of the clinical severity and/or the age at diagnosis. The acute form occurs early in
infancy and causes severe liver damage leading to liver failure and
death. The chronic form manifests itself later in infancy or childhood
with symptoms such as progressive liver cirrhosis, renal tubular
dysfunction, and a high incidence of hepatocellular carcinoma (8). HT1
is the most severe of the diseases associated with the enzymes of the
tyrosine catabolic pathway. Although its prevalence worldwide is low
(1:120,000 births), it shows a high incidence in some populations such
as that of the Saguenay-Lac-St-Jean region (Canada), where it affects
1:1,846 newborns indicating a carrier frequency of 1:20 inhabitants
(9). The high incidence of HT1 in this region is presumably the result
of a founder effect involving mostly the IVS12+5g
a splice mutation
(10, 11).
The human FAH gene is localized to the q23-q25 region of chromosome 15 (12), contains 14 exons, and covers ~35 kilobases of DNA (13, 14). At
this time, 34 mutations have been reported (8, 15, 16). These include
18 missense mutations, 10 splice mutations, 5 nonsense mutations, and 1 silent mutation. These mutations are evenly spread along the FAH gene
but with a slightly higher frequency in some parts of exons 8 and 13.
The human FAH enzyme has been purified to homogeneity (2, 17), and the
crystal structure of recombinant mouse FAH has recently been reported
(18). FAH represents a new class of metalloenzymes that possess a
unique
/
fold. The crystal structure of FAH and its active site
should prove particularly helpful in understanding the effects of
mutations on both the structure and the activity of the enzyme.
To determine the effects of missense mutations on the structure and
activity of FAH, we used site-directed mutagenesis to generate mutant
FAHs and examined the expression and enzymatic activity of mutant
proteins in a bacterial expression system and by transient expression
after transfection in mammalian cells. Circular dichroism spectra were
measured for the wild-type FAH and four variants containing
HT1-associated amino acid substitutions, and structural descriptions of
HT1-associated amino acid substitutions were made based on crystal
structure of murine FAH (18). Eight missense mutations (all previously
reported in HT1 patients) were analyzed: N16I (19), F62C (14), A134D
(13, 20, 21), C193R (22), D233V (20, 23), W234G (24), Q279R (25), and
R341W, the so-called pseudo-deficiency mutation (26, 27). There is
still no clear relation between the genotype and the phenotype in HT1,
which varies from an acute to a more chronic form. Mutations analyzed
in this study were chosen in a manner to include mutations affecting
residues in different parts of the FAH structure and involving mostly
residues conserved from Caenorhabditis elegans to Homo
sapiens. Some of the mutations were studied for specific reasons.
For example, the C193R mutation was analyzed to determine whether the
cysteine residue at position 193 was essential to the structural
integrity of the enzyme. Other mutations were examined because they
were located in the enzyme's active site (D233V and W234G). The
molecular basis of the R341W mutation, described as a pseudo-deficiency
mutation, was investigated because of the unusual phenotype observed in
homozygote individuals who show no symptoms of the disease. Finally,
the Q279R mutation was studied because it represented a new mutation
for which the molecular basis had not yet been described. Many of the
mutated proteins were found to be inactive, probably as a result of
misfolding of the mutated FAH.
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EXPERIMENTAL PROCEDURES |
FAH Cloning in the pET30 Vector--
A human FAH cDNA clone
was obtained by amplification of cDNA reverse-transcribed from
mRNA of a normal liver (patient 8688). The amplification reaction
mixture (50 µl) contained 5 µl of cDNA, 5 µl of 10× PCR
buffer (Expand Long Template PCR System, Roche Molecular Biochemicals),
500 µM dNTPs, 200 ng of primers TANR130 and RT025, and
2.5 units of recombinant Taq-Pwo polymerase
(Roche Diagnostics). TANR130 primer (5'-CAT GTC CTT CAT CCC GGT GGC-3') is situated at the very beginning of the 5'-coding end of FAH cDNA,
a cytosine has been added in front of the start ATG codon of the FAH
gene. RT025 (5'-GGG AAT TCT GTC ACT GAA TGG CGG AC-3') is located in
the 3'-noncoding end of the gene. The 5' extremity of RT025 is not
complementary to the cDNA but contains an EcoRI restriction site used to clone the amplification product in a vector.
The reaction mixture was covered with 50 µl of mineral oil and
incubated at 95 °C for 5 min, 53 °C for 5 min, and 72 °C for
40 min to allow the synthesis of the second strand of the cDNA. The
PCR performed on a DNA Thermal Cycler (model N801-0150, PerkinElmer
Life Sciences) included 35 cycles of the following program: 40 s
at 95 °C, 1 min at 53 °C, and 2 min at 72 °C. A final
extension of 15 min at 72 °C was done to complete the elongation. The amplification product was digested with NcoI (2,700 units/ml, Amersham Pharmacia Biotech) and blunted with nuclease S1
(343,400 units/ml, Amersham Pharmacia Biotech). After this first
enzymatic digestion, the product was digested with EcoRI to
obtain a cohesive end at the 3' extremity. The same enzymatic
digestions were performed on the pET30 vector (Novagene). The digested
amplification product was then ligated to the pET30 vector using the T4
DNA ligase (400,000 units/ml, New England Biolabs), and the pET30FAH
construction was fully sequenced on an ABI 373XL.
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed on the pET30FAH vector using the Quick ChangeTM
site-directed mutagenesis kit (Stratagene). PCR reactions (50 µl)
contained 10 mM KCl, 10 mM
(NH4)2SO4, 20 mM
Tris-HCl (pH 8.8), 2 mM MgSO4, 0.1%
Triton® X-100, 0.1 mg/ml nuclease-free bovine serum
albumin, 50 ng of pET30FAH vector, 125 ng of each complementary primer
(Table I), 250 µM of each
dNTP, and 2.5 units of Pfu DNA polymerase. The PCR
conditions used were 30 s at 95 °C for DNA melting, followed by
12 cycles of elongation (each cycle consisting of 30 s at
95 °C, 1 min at 55 °C, and 13.5 min at 68 °C). The DNA in the
reaction mixtures was digested 1 h at 37 °C using 10 units of
DpnI (Stratagene) and was used to transform competent
bacterial cells. Clones containing the site-directed mutations were
verified by sequencing (results not shown) to ensure that no other
mutations were present in the gene.
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Table I
PCR primers and their location in the FAH gene
The mutated nucleotides are in bold lowercase letters. nt, nucleotides.
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FAH Cloning in pCEP4 Mammalian Expression Vector--
The
wild-type and mutated pCEP4FAH mammalian expression vectors were
constructed by inserting the fragments of pET30FAH vectors digested
with KpnI (10,000 units/ml, New England Biolabs) and HindIII (20,000 units/ml, New England Biolabs) and
containing the wild-type or mutant FAH gene into
KpnI-HindIII-cut pCEP4 vector (Invitrogen). The
fah fragments were ligated to digested pCEP4 vector using T4
DNA ligase (40,000 units/ml, New England Biolabs).
Preparation of Samples for pET30FAH Expression in Bacterial Cells
and Purification--
The wild-type vector pET30FAH and mutant vectors
were transformed in the GJ1158 strain of Escherichia coli.
The cells were grown to an A600 of 0.4-0.5 at
37 °C in Luria-Bertani medium with NaCl omitted, containing 10 g of tryptone and 5 g of yeast extract per liter (pH 7.0), with 50 µg/ml kanamycin. The temperature was then lowered to 30 °C, 0.3 M NaCl was added to the medium, and cultures were further
incubated for 5 h (28). Cells were harvested and pellets
resuspended in 20 ml of 10 mM phosphate buffer (pH 7.3).
Each sample was introduced into a French press cell and lysed at
800 p.s.i. of pressure. Afterward, samples were centrifuged at
10,000 × g for 30 min at 4 °C. Following this
centrifugation, the soluble supernatant was recovered and protein
concentration was determined using the Bio-Rad protein assay. For CD
measurements, the recombinant proteins were purified by affinity
chromatography through their His tag on nickel-nitrilotriacetic acid
superflow columns (Qiagen).
CD Measurements--
Samples for CD measurements were dialyzed
overnight against a 1000-fold excess volume of 10 mM sodium
phosphate (pH 7.0) at 4 °C. Wild-type FAH remains soluble in excess
of 1 mg/ml under these conditions; however, precipitation was noted for
several of the variants and was removed by centrifugation at
14,000 × g. The yield of soluble N16I, F62C, and W234G from the
bacterial expression system was not sufficient for making CD
measurements. Concentrations of supernatants of the dialyzed samples
were determined by scanning UV absorbance spectra using an extinction
coefficient of 1.3 ml·mg
1·cm
1 at
280 nm. Less than 3% of the C193R sample remained soluble following
dialysis, preventing further CD analysis of this sample. Samples were
diluted to 0.12 mg/ml, and CD spectra were recorded as an average of 12 scans at room temperature using a Jasco J720 instrument and a 1-mm
light path. Base-line spectra were recorded using individual dialysis
solutions as a blank for each sample. Mean residue ellipticities were
calculated using a mean residue weight of 110. Final protein
concentrations were calculated using photomultiplier tube voltages and
standard curves based on serial dilutions of wild-type FAH. Data
smoothing has not been performed. Protein concentration-dependent
differences between the CD spectra were assessed based on the
proportionality between variant and the wild-type spectra in the
240-190 nm range, and by calculating the ratio of ellipticities
measured at fixed wavelengths (data not shown) and measured at two
minima apparent in the spectra (Fig. 2 and Table IV). Differences
between the wild-type and Q279R spectra were judged to be
insignificant, based on these criteria.
Expression in Mammalian Cells--
CV-1 (kidney African Green
monkey cells, American Type Culture Collection) were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (Medicorp Inc.) at 37 °C
with 5% CO2. The day prior to transfection, 2 × 106 CV-1 cells were seeded in a 75-cm2 flask
and grown at 37 °C. The cells were transfected with 2 µg of
pCEP4FAH wild-type or mutant DNA using the FuGENE6 kit (Roche Molecular
Biochemicals) according to the manufacturer's recommendations. Six
hours after transfection, cells were washed twice in PBS and fresh
DMEM, 10% fetal bovine serum medium was added to the cells. Forty
hours after transfection, cells were washed in PBS, trypsinized and
resuspended in 10 mM phosphate buffer (pH 7.3) for
homogenization using a Teflon pestle and a syringe. The homogenized
extract was centrifuged 15 min at 10,000 × g, both the
supernatant and the pellet were recovered, and protein concentration
was determined using the Bio-Rad protein assay. These samples were used
for kinetics assays as well as for Western blot analysis.
Nontransfected CV-1 cells showed no FAH activity (data not shown).
FAH Activity Assay and Western Blot Analysis--
FAH hydrolytic
activity was determined as described previously using about 50 µM FAA as a substrate (2). Briefly, the decrease of
absorbance at 330 nm, which corresponds to hydrolysis of the FAA
substrate, was measured at room temperature using a spectrophotometer (Ultrospec III (Amersham Pharmacia Biotech) or Cary Varian 100) for the
CV-1 cell extracts (using 40-150 µg of total protein/assay) and the
bacterial extracts (using 5-10 µg of total protein/assay). To
correct for differences in transfection efficiency and expression of
the mutant FAH proteins, the hydrolytic activity of FAH against FAA was
expressed as nanomoles of FAA hydrolyzed/min × mg of FAH, as
determined by the following equation.
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(Eq. 1)
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1.35 µM
1
cm
1 was used as the extinction coefficient of
FAA at 330 nm, and
OD represents the change in optical density at
330 nm/min.
The amount of FAH was measured by a quantitative Western blot assay.
The cell extracts were loaded on a 12% SDS-PAGE gel along with known
quantities (0.1, 0.2, 0.5, and 1 µg) of purified recombinant human
FAH protein. Proteins were transferred to an Immobilon-P membrane
(Millipore) for Western blot analysis (29). The primary antibody,
anti-FAH (antibody 488) (2) was used at a 1:25,000 dilution, whereas
the secondary antibody, alkaline phosphatase coupled to a goat
anti-rabbit IgG (Jackson Immunoresearch Laboratories), was diluted
1:50,000. A standard curve showing the integrated density of the signal
on the quantity of FAH present in the sample was obtained from NIH
Image program analysis by measuring the signal intensity of FAH
containing samples on films.
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RESULTS |
Characterization of Mutant FAH Proteins by Expression in Bacterial
Cells--
To study the structure-function of FAH, various missense
mutations found in the FAH gene of different HT1 patients (Table II) were introduced in a human FAH
cDNA by site-directed mutagenesis. These patients were either
homoallelic or heteroallelic and exhibited different phenotypes ranging
from normal to an acute form. For example, the patient heteroallelic
for the Q279R and the IVS6
1g
t mutations showed mild clinical
symptoms until 36 years but then developed hepatocellular carcinoma
(25).
The mutated FAH constructs were introduced in E. coli and
the corresponding proteins induced with NaCl (28). The soluble recombinant protein fractions were analyzed by Western blot and for
enzymatic activity. As shown in Fig.
1A, all mutated FAH proteins with the exception of N16I, F62C, and W234G were expressed in the
soluble fraction of GJ1158 E. coli strain. The FAH harboring N16I, F62C, and W234G were expressed but were retained in the insoluble
cell fraction (data not shown). Next, the hydrolytic activity of the
mutated FAH proteins was measured using FAA as the substrate. As shown
in Table III, all mutated FAHs with the exception of R341W and Q279R
were inactive in this hydrolytic assay. R341W and Q279R showed
activities equal to that of normal FAH.

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Fig. 1.
Analysis of FAH expression by Western
blot. Figure shows Western blot analysis of wild-type FAH and
mutant FAH protein expression in the cell-soluble fraction of E. coli cells (A) and in CV-1 transiently transfected
cells (B). The mammalian cells were fractionated in a
10,000 × g insoluble fraction (B,
top, 20 µg) and a supernatant-soluble fraction
(B, bottom, 5 µg). Samples from cellular
extracts were loaded on the gel as follows: pork liver (5 µg),
wild-type FAH, N16I, F62C, A134D, C193R, D233V, W234G, Q279R, and
R341W.
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Characterization of Mutant FAH Proteins by Transient Expression in
CV-1 Cells--
To check if these mutated proteins were equally
expressed in a mammalian background, the FAH coding fragment of each
pET30FAH construct was subcloned in the pCEP4 mammalian expression
vector and transfected in simian CV-1 cells. Forty-eight hours after transfection, cells were homogenized and proteins from the soluble and
insoluble fractions (10,000 × g pellet) were loaded on
a 12% SDS-PAGE gel and immunoblotted with the anti-FAH antibody. As can be seen in Fig. 1B, all mutant proteins were expressed
in CV-1 cells. However, proteins containing the N16I, F62C, C193R, and
W234G mutations were only present in the insoluble fraction of the
cellular extract (Fig. 1B, top), which suggests
that these proteins are subject to misfolding and aggregation in
mammalian CV-1 cells. This would explain their absence from the soluble fraction of cellular extracts. To assess whether the expressed proteins
were localized properly in the cell, immunofluorescent staining with
anti-FAH was performed on CV-1 cells 48 h after transfection. The
mutated proteins found in the insoluble fraction of the cellular
extracts by Western blot analysis (N16I, F62C, C193R, and W234G) were
shown to form aggregates mainly in the perinuclear-Golgi region of the
cell (data not shown). This observation supports the hypothesis that
the presence of these mutations interferes with the proper folding of
the enzyme. The other FAH variants (A134D, D233V, Q279R, and R341W)
were evenly distributed in the cytoplasm, much like the wild-type
protein (data not shown).
Assay of the hydrolytic activity of the mutant FAHs in transfected
mammalian cells gave results identical to those seen for proteins
expressed in bacteria, i.e. only the R341W and Q279R proteins had hydrolytic activity (Table
III), which was similar to that of the
wild-type enzyme. The specific activity of FAH expressed in bacterial
cells is notably much higher than that measured in transfected
mammalian CV-1 cells. The activity in these cells is comparable to that
measured in pork liver (780 nmol·min
1·mg
1
FAH).
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Table III
Enzymatic activity of wild-type and mutated FAH
FAH hydrolytic activity was determined as described in the experimental
section. Data are means ± S.D. (n = 3).
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Effects of Mutations on FAH Structure--
To assess structural
perturbations caused by HT1-associated amino acid substitutions,
circular dichroism (CD) spectra were measured for wild-type FAH and the
A134D, D233V, Q279R, and R341W variants. Representative spectra are
shown for wild-type FAH, FAH D233V, and FAH A134D in Fig.
2. The wild-type FAH CD spectrum is
characterized by minima at 225.0 and 208.8 nm, a crossover point at
201.9 nm, and a maximum at 196.0 nm. This spectrum is consistent with
an
/
structure in solution. The FAH crystal structure contains
27%
-strand and 18%
-helical secondary structure (18).
Significant differences were observed with all the HT1 variants, except
Q279R (Fig. 2 and Table IV). The largest
differences involved the position of the minimum between 220 and 225 nm
and the intensity of the CD bands across the spectrum. The spectrum for
A134D, having a minimum at 220.0 nm, enhanced negative ellipticity between 220 and 200 nm, and diminished positive ellipticity below 200 nm, deviated most from the wild-type spectrum (Fig. 2). The R341W FAH
spectrum shows enhanced negative ellipticity above 201 nm and
diminished positive ellipticity below 201 nm, relative to wild-type.
D233V FAH also showed enhanced negative ellipticity above 201 nm, but
nearly identical ellipticity below 201 nm, relative to wild-type. As
the far UV CD spectrum of a protein is primarily due to the environment
of the peptide chromophore (30), differences in the CD spectra of the
FAH variants relative to wild-type FAH are consistent with small
changes in secondary structure. Spectra for N16I, F62C, C193R, and
W234G could not be accurately measured due to problems with
precipitation and solubility (see "Experimental Procedures"). Thus,
all of the HT1-associated amino acid substitutions studied here, with
the exception of Q279R, have effects on the FAH structure in
vitro as indicated by altered CD spectra and/or reduced
solubility. These structural perturbations may, in turn, account for
the functional loss of FAH activity in individuals carrying these
HT1-associated amino acid replacements. Structural representation of
FAH and effects of these different mutations are shown in Fig.
3 (see "Discussion").

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Fig. 2.
CD spectra of FAH. The far UV CD spectra
for wild-type FAH (thick line), D233V FAH
(thin line), and A134D FAH (dashed
line) are shown between 190 and 270 nm. Mean residue
ellipticity is in units of
degrees·cm2·dmol 1.
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Table IV
Major features in CD spectra
The positions of the major features present in the indicated CD spectra
were identified using the Jascow Standard Analysis Program version
1.20. Min1:Min2 is the ratio of the minimal mean residue ellipticity
values measured at the wavelengths indicated in columns two and three.
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Fig. 3.
Structural representation of FAH
(A) and mutations (B-E).
A shows the dimeric structure of FAH. The effects of
the mutations are shown in B-E (B, N16I;
C, F62C; D, C193R; E, D233V and
W234G). Carbon atoms are in yellow, nitrogen atoms in
blue, oxygen atoms in red, sulfur atoms in
green, Ca2+ in purple, and
Mg2+ in gray; black lines
represent hydrogen atoms.
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DISCUSSION |
Using site-directed mutagenesis, we generated a number of
naturally occurring missense mutations detected in the FAH gene of HT1
patients to try to understand the structure-function relationship of
FAH. Although some of these mutations had previously been tested in an
in vitro translation system (13, 14, 19, 21, 23, 24, 27), no
studies examining the properties of the mutated proteins in a bacterial
and in a mammalian background have been reported.
The HT1-associated amino acid substitutions studied here can be grouped
into two classes based on the solvent inaccessibility of the altered
residue. Nonconservative substitutions involving solvent-inaccessible residues are more likely to have adverse steric
effects and are less likely to be tolerated within enzyme structures
than solvent-accessible residues. Indeed, the FAH substitutions N16I,
F62C, C193R, and W234G appear to cause gross structural misfolding
and/or instability that results in aggregation and precipitation when
expressed in either mammalian or bacterial cells. Phaneuf et
al. (19) identified N16I as the first causal mutation in FAH
responsible for HT1. After transfecting the mutant cDNA in CV-1
cells, they reported that these cells produced FAH mRNA but
FAH was not detected by Western blot analysis nor was any FAH activity
detected. They suggested that the mutated protein was unstable. The
present data show that the N16I protein is produced in bacterial cells
and in transfected mammalian CV-1 cells but that it is insoluble. In
fact, the Asn-16 side chain forms hydrogen bonds with main chain atoms
of both the N- and C-terminal domains (Fig. 3 B). The N16I
substitution disrupts these electrostatic contacts between domains and
the bulkier Ile side chain could cause steric problems between the
domains, which would explain the gross structural impairment we observe
for FAH N16I.
Awata et al. (14), who first documented the F62C mutation of
FAH, had demonstrated that this mutation was responsible for the
decreased activity of the enzyme in a patient. The present study agrees
with their data. Thus, the Phe-62 side chain stacks between the Phe-50
and Phe-70 side chains to form a hydrophobic core in the N-terminal
domain (Fig. 3C). The F62C substitution introduces a cavity into this
hydrophobic core. This effect of the mutation on the structure of the
enzyme causes its retention as aggregates in the perinuclear-Golgi
region of the cell.
The C193R mutation (22), also disrupts the structure of the protein
since it forms insoluble aggregates mainly localized in the
perinuclear-Golgi region of the cell. The Cys-193 side chain is located
in a C-terminal hydrophobic core (Fig. 3D) in van der Waals
contact with the
-helix that contributes the Arg-237 and Gln-240
side chains to the FAH oxyanion hole. A conservative C193S substitution
is found in the wild-type rat FAH. The C193R substitution would
introduce unfavorable steric contacts in this region and might also
alter the conformation of these two critical active site residues. This
would explain the loss of activity that we observed.
The W234G mutation was reported in an American HT1 patient compound for
this mutation and the IVS12+5g
a splice mutation (24). We observed
that the mutated protein showed no FAH activity and was enriched in a
cell-insoluble fraction. Trp-234 is located adjacent to the metal ion
binding residue Asp-233 (described below). In addition to affecting the
binding of two metal ions, the W234G substitution introduces a large
cavity in a hydrophobic core (Fig. 3E). Thus, substitutions
of the solvent-inaccessible residues described above (N16I, F62C,
C193R, and W234G) result in gross structural defects associated with
the disruption of native structural interactions or by the introduction
of cavities and bad steric contacts.
Two exceptions to the gross structural perturbations described above
are the A134D and D233V substitutions. Immunofluorescent staining of
A134D FAH shows that the protein is evenly distributed throughout the
cytoplasm, which suggests that this mutation does not severely impair
its structure (data not shown). Ala-134 is located in a hydrophobic
environment adjacent to the His-133 catalytic base and also near
Tyr-128 at the entrance of the active site (18). The A134D substitution
introduces a charged residue and bad steric contacts into this
hydrophobic environment, presumably affecting substrate binding and
catalysis by perturbing the conformation of the active site. Patients
homozygous for the D233V mutation have a normal amount of liver FAH
mRNA but a reduced amount of immunoreactive material and no FAH
activity (23). In the present study, Western blot analysis as well as
immunofluorescent staining show a comparable level of expression of
D233V-substituted and wild-type FAH. The mutated enzyme showed no
hydrolytic activity either when expressed in bacteria or in mammalian
cells, a finding similar to measurements in liver extracts of patients
reported by Rootwelt et al. (23). The Asp-233 side chain is
a ligand for the catalytic metal ion and binds a second metal ion that stabilizes the conformation of a loop between residue Lys-253 of the
FAH oxyanion hole and Thr-257 (Fig. 2E). The D233V mutation introduces a hydrophobic group into a charged environment and presumably disrupts two metal ion binding sites and the conformation of
important catalytic residues. It is surprising that significant amounts
of soluble protein are produced in mammalian and bacterial systems for
both of these variants. Loss of metal ion binding in the active site
would be expected to cause charge repulsion of the acidic side chain
ligands of the metal ions. The CD spectra for A134D differs most from
the wild-type FAH spectrum (Fig. 2 and Table IV), indicating that
substantial structural changes are associated with this substitution.
Differences between the D233V and wild-type CD spectra were less
pronounced, but still consistent with minor structural perturbations
(Table IV). Although A134D and D233V involve residues that are
inaccessible to bulk solvent in the surrounding solution, Ala-134 and
Asp-233 are in close proximity to ordered water molecules bound in the
FAH active site. The FAH active site shows considerable flexibility
when different crystal structures are
compared.2 Therefore, limited
conformational changes associated with these substitutions appear to be
tolerated within the flexible FAH active site.
The Q279R and R341W substitutions affecting solvent accessible side
chains had no significant effect on enzymatic activity. The Q279R
mutation was reported in a compound heterozygote patient (Q279R/IVS6-1g
t), who is unusual in the sense that she represents a rare case of HT1 to have lived over 30 years of age and showed few
symptoms associated with the disease until she developed hepatocellular carcinoma (25). Site-directed mutagenesis and expression of Q279R in
E. coli cells and transiently transfected CV-1 cells show
that the catalytic activity of the mutated protein is similar to that
of the wild-type protein and a normal localization pattern of
expression. The Gln-279 side chain forms two hydrogen bonds with the
Gln-328 side chain and a single hydrogen bond with the Tyr-323 main
chain oxygen atom in a solvent-accessible loop. Although these
interactions are likely to be disrupted by the Q279R substitution, solvent accessibility in this region is likely to tolerate this amino
acid replacement by allowing the Arg side chain to rotate out into the
surrounding solution, or by allowing slight alteration of the loop
conformation to enable the Arg side chain to participate in hydrogen
bonding similar to the Gln-279 side chain. In fact, no structural
perturbation was indicated by the CD spectrum for Q279R relative to
wild-type FAH. Therefore, the present study shows no direct effect of
the Q279R mutation on the structure or activity of the enzyme. However
since the Q279R mutation is located in the 5' donor splice region of
exon 9, we believe that this mutation acts as a splice mutation
in vivo, which would explain the HT1 phenotype of the
patient.3
The last mutation analyzed is R341W described as a pseudo-deficiency
mutation because patients homozygous for the mutation show no
pathological symptoms of the disease (26). Northern blot analysis
showed a normal amount of FAH mRNA in fibroblasts from homozygous
patients but very little immunoreactive FAH (27). Measurement of
enzymatic activity of the mutated FAH in an in vitro protein
synthesis system showed a reduced activity on the order of 25-30% of
that of normal FAH (27). This was higher than the activity measured in
fibroblasts leading to the suggestion that the R341W protein was more
rapidly degraded in vivo than in vitro.
In the present study, the R341W protein had a specific activity very
similar to that of wild-type FAH when expressed either in bacteria or
in CV-1 cells. Structural data indicate that the Arg-341 side chain
protrudes into a water-filled space near the FAH dimer interface and
makes van der Waals contacts with the Ser-164 side chain of the
opposite subunit. The solvent accessibility of these residues suggests
that this interaction is not very significant and similar contacts
could be made by the Trp side chain in the R341W substituted protein.
Despite having good enzymatic activity (Table III), it is interesting
to note that the R341W spectrum was altered relative to wild-type FAH
(Table IV). Reduced enzymatic activity of the pseudo-deficiency
phenotype associated with R341W has been suggested to result from
decreased stability or increased turnover in patient fibroblasts (27).
Although the studies presented here do not address enzymatic half-life
within mammalian cells, it is conceivable that the perturbation of the
FAH structure caused by substituting the charged surface Arg side chain
with an indole ring is sufficient to account for a decreased enzyme stability.
In summary, three kinds of mutations in the FAH gene were
characterized: 1) mutations resulting in gross structural perturbations (N16I, F62C, C193R, and W234G), 2) mutations causing limited
conformational changes tolerated within the flexible active site (A134D
and D233V), and 3) mutations that display no significant effect on
enzymatic activity (Q279R and R341W). The availability of the crystal
structure of FAH can provide helpful hints in predicting and testing
the effects of newly discovered mutations by their localization within the enzyme's tertiary structure and sheds new light on the
structure-function relationship of the enzyme.