From the aUnit of Biochemistry and Molecular Biology, International University of Catalonia, 08190 Sant Cugat del Vallés, bBioinformatics Laboratory (CAB-CSIC), 28850 Torrejón de Ardoz, Madrid, cDepartment of Pharmacology and Physiology, University of Zaragoza, 50009 Zaragoza, eProtein Design Group (CNB-CSIC), Cantoblanco, 28049 Madrid, the gDepartment of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain, hWillink Biochemical Genetic Unit, Manchester M27 4HA, United Kingdom, the iDepartment of Pediatrics, Leicester General Hospital, Leicester LE5 4PW, United Kingdom, jFundación para el Estudio de las Enfermedades Neurometabólicas, 1425 Buenos Aires, Argentina, and kDivision of Metabolic and Endocrine Diseases, University Children's Hospital, D-69120 Heidelberg, Germany
Received for publication, April 23, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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The origin of the disease is a mutation in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)1 lyase (HL), which cleaves HMG-CoA to form acetyl-CoA and acetoacetate. This is the final step in the ketogenic pathway and leucine catabolism. The gene was assigned to the distal short arm of human chromosome 1p36.1 (5). It contains 9 exons and is transcribed in an mRNA of 1.7 kb (6). The presence of the protein inside mitochondria and peroxisomes has been reported. The active site of HL has been shown to include the residues Cys266 and His233 (7, 8). To date, 38 probands have been diagnosed at the molecular level, and 22 different mutations of the HL gene have been reported (5, 6, 919). Only one of the missense mutations described, H233R, directly affects the catalytic residues. In contrast, there is no explanation for the effect of the other five reported missense mutations in the functional structure of the enzyme.
Here we report three novel homozygous missense mutations affecting patients
from Germany, England, and Argentina, and we describe the effect of these
mutations in the protein activity by expression studies in Escherichia
coli. We also propose a three-dimensional model for the human HMG-CoA
lyase containing a ()8 barrel structure, usually
called TIM barrel, predicted by the relationship between HMGL-like
and hisA (1qo2
[PDB]
) family proteins. All the missense mutations reported
to date are located in the proposed structural model around the catalytic
cavity, and their possible effects on enzyme activity are discussed.
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MATERIALS AND METHODS |
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The first symptoms of the English proband (B. P.) appeared at 48 h of age, and tests revealed metabolic acidosis (high pyruvate and lactate), hypoglycemia (1.2 mM), and 13.9 mM bicarbonate. The organic acids (3-hydroxyisovalerate, 3-methylglutarate, 3-methylglutaconate, glutarate, adipate, and other dicarboxylic acids) found in blood and urine supported the diagnosis of 3-hydroxymethylglutaric aciduria. HMG-CoA lyase activity was 0.
Aciduria in the German patient (T. J.) first appeared at 4 months of age after DPT/HiB/polio immunization, with recurrent vomiting, muscular hypotonia, and comatose attacks. He showed hyperammonemia, liver dysfunction, and massive excretion of organic acids typical of HMG-CoA lyase deficiency. Enzyme activity was 0.2 nmol·min1·mg1 protein. The boy recovered well with intravenous glucose and at 8 years shows no mental retardation nor renal, hepatic, or cardiac abnormalities.
Fibroblast Culture and DNA IsolationA skin biopsy was taken from the patient B. P. Fibroblast explants were cultured in 25-cm2 flasks in Dulbecco's modified Eagle's medium, 100 IU/ml penicillin, and 100 mg/ml streptomycin, 2 mM glutamine, 10% fetal calf serum, and 95% air, 5% CO2. Mutational analysis was performed in DNA isolated from leukocytes (D. S. and T. J.) or cultured fibroblasts (B. P.). DNA was isolated using the DNAzol® Reagent kit from Invitrogen.
PCR Amplification of Genomic DNATo amplify all exons, 200 ng of genomic DNA was amplified in a 100-µl mixture containing 0.2 mM of each dNTP, 25 pmol of each primer, 2.5 units of Taq DNA polymerase in 1x PCR buffer, and 2 mM MgCl2 and the primers flanking intron sequences (11). The conditions used are as follows: 98 °C for 5 min and subsequently 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s for 35 cycles, and finally 7 min at 72 °C.
Missense Mutation Restriction AnalysisTwo specific primers, mut201 and mut204, were designed to introduce an individual nucleotide mutation, which would generate a new restriction site NcoI or DraII, respectively, in the control DNA. The mutations S201Y (c602C>A) and D204N (c610G>A) abolished the restriction sites introduced by the primers mut201 and mut204, respectively. The mutation S75R (c225C>G) abolished an AluI site, present in the control DNA. Primers used in the amplifications are shown as follows. The modified nucleotide is underlined. For the Mut201 sequence primer, 5'-GGGGTGCCCACACCAATGGTGTCCCCCATG-3'; for the Mut204 sequence primer, 5'-TGATCCCTGGGGTGCCCACACCAATGGGG-3'; and for the I3R1 sequence primer, 5'-CCTATGTTCTCAACTTCTAC-3'.
Genomic fragments amplified with primers F7E (18) and Mut201, with primers F7E and Mut204, or with primers F1 (15) and I3R1 in controls and patients were used as substrates for analytical digestion with NcoI, DraII, or AluI, respectively. Five units of restriction enzymes were added to 20 µl of reaction medium. Digestions were incubated for 3 h at 37 °C; the resulting restriction fragments were fractionated on a 4.5% metaphor-agarose gel.
Construction of Expression PlasmidsHMG-CoA lyase cDNA was amplified by reverse transcriptase-PCR from 1 µg of total RNA isolated from control human fibroblasts, using Superscript II kit (Amersham Biosciences), as described by the manufacturer. The region of the cDNA coding for the mature protein plus 5' Met and Gly codons was amplified with Pfu DNA polymerase (Stratagene) and primers that introduced an NcoI site (5'-GCACCTCATCCATGGGCACT-3') and a BamHI site (5'-CCCCAGGGATCCAGGTGGGC-3') at the ends of the amplified fragment. The 950-bp PCR fragment was cloned into NcoI/BamHI-cut pTrc99A plasmid to give the expression plasmid pTr-HLwt. The insert was sequenced with Applied Biosystems 373 automated DNA sequencer to verify that no mutations had been introduced during PCR amplification.
Mutants S75R, S201Y, and D204N of HL were constructed using the "Quick Change" PCR-based mutagenesis procedure (Stratagene) with the pTr-HL-wt plasmid as a template. Primer 5'-TATAGAAACCACCAGGTTTGTGTCTCCTAAG-3' was used to construct pTr-HL-75R; primer 5'-GCTGCTACGAGATCTACCTGGGGGACACCATTG-3' was used to construct pTr-HL-201Y; and primer 5'-GAGATCTCCCTGGGGAACACCATTGGTGTGG-3' was used to construct pTr-HL-204N. The appropriate substitutions and the absence of unwanted mutations were confirmed by sequencing the inserts.
Expression of HL in E. coliE. coli JM105 cells transformed
with the expression plasmids were grown in LB broth supplemented with
ampicillin (50 µg/ml) at 25 °C. When A600 = 0.6,
the expression of HL was induced by addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 1 mM. After 6 h of induction, cells were recovered
by centrifugation at 5,000 x g for 15 min at 4 °C and
frozen to 70 °C until required for use.
Cell pellets were resuspended in lysis buffer (NaCl 50 mM, Tris-HCl, pH 7.5, 5 mM, DNase 10 µg/ml, RNase 10 µg/ml, phenylmethylsulfonyl fluoride 0.6 mM, pepstatin 1 µg/ml, leupeptin 2 µg/ml, lysozyme 1 mg/ml) and incubated for 15 min at 4 °C. Afterward, the tubes were frozen to 70 °C and thawed to 37 °C. Soluble proteins were recovered by centrifugation at 10,000 x g for 10 min at 4 °C and immediately frozen and stored at 70 °C. Protein quantification was performed using the Bio-Rad protein assay with bovine albumin as standard.
HMG-CoA Lyase ActivityHMG-CoA lyase activity was measured by simple spectrophotometric method that determines the amount of acetoacetate produced (20). For HL-specific activity determination, 2 µg of soluble protein extracts and 800 nmol of substrate (HMG-CoA) were used. In the case of mutation extracts, determinations were also carried out with 20 or 75 µg of soluble protein extract. The reaction assay was performed at 37 °C for 15 min in a final volume of 250 µl. One enzyme unit represents the formation of 1 µmol of acetoacetate in 1 min. For Km determinations, 2 µg of soluble crude extract was used. HMG-CoA concentrations ranged from 400 to 6400 µM. Results are given as mean values of at least four independent experiments.
Western Blot AnalysesA polyclonal rabbit antibody against residues 307321 of human HL (which recognizes the COOH terminus of the enzyme) was raised by Sigma-Genosys. 50 µg of E. coli protein extract was subjected to SDS-PAGE. Electroblotting to nitrocellulose sheets was carried out for 2 h at 120 mA. Immunodetection of HL was performed using the antibody anti-HL (dilution 1:10,000), and the blots were developed with the ECL Western blotting system from Amersham Biosciences.
Prediction of a Structural Model for HMGL_HUMANThe multiple sequence alignment used to provide the anchor points of the modeled human 3-hydroxy-3-methylglutaryl-CoA lyase (HMGL_HUMAN) to the template three-dimensional structure 1qo2 [PDB] (hisA (21)) was prepared by aligning the HMGL_like-PF00682- and His_biosynth-PF00977-Pfam data base families (22). This alignment is similar to that obtained elsewhere (23) after four interactions of a PSI-Blast search of the NR protein sequence data base from the NCBI, using the amino acid sequence of 1qo2 [PDB] as seed (24).
To obtain a three-dimensional model of HMGL_HUMAN (amino acids
36287), the program Swiss-Pdb Viewer and the SWISS-MODEL server
facilities
(2528)
(www.expasy.ch/swissmod/SWISS-MODEL.html)
were used. The atomic coordinates of the ()8 barrel
template structure used to model the protein were obtained from the Protein
Data Bank (PDB)
(www.rcsb.org/pdb;
entry 1qo2
[PDB]
). The atomic coordinates of the 3-hydroxy-3-methylglutaryl-CoA
molecule were obtained from the PDB entry 1qax
[PDB]
. All missense mutants described
in the study were modeled using the HMGL_HUMAN model as template.
The quality of the HMGL_HUMAN model was checked using the programs ProsaII (Center of Applied Molecular Engineering, Salzburg, Austria (29); the WHAT-CHECK routines (30) from the WHAT IF program (31) and the PROCHECK validation program from the SWISS-MODEL server facilities (32). The comparison of the values from the template structure 1qo2 [PDB] versus the proposed HMGL_HUMAN model is accessible through the URL, www.cnb.uam.es/~pagomez/HM_Check.html. Briefly, the overall quality values of the model are poorer than the usual ones for experimental x-ray or NMR structures but are acceptable in the expected region for protein structure models.
The interaction between HMG-CoA as the substrate and the proposed model was calculated using the macromolecular docking program "Hex" (33) based on spherical polar Fourier correlations. The picture of the surface, section, and electrostatic potentials for the proposed protein models was produced using the GRASP program (34), and the ribbon plots were drawn with RASMOL (35).
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RESULTS |
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Residues affected by mutation presented a high degree of conservation compared with HL from other species. Asp204 is invariant in the various HMG-CoA lyases from mammals (human, bovine, rat, and mouse), chicken, plants (Arabidopsis thaliana, and rice), and bacteria (Pseudomonas mevalonii, Bacillus subtillis, and Rhodospirillum rubrum) for which primary structures have been elucidated. Moreover, Asp204 is also invariant in 59 enzymes whose mechanism of reaction is analogous to HMG-CoA lyase, such as homocitrate synthase, 2-isopropylmalate synthase, oxovalerate aldolase, and pyruvate carboxylase from mammals, yeast, bacteria, and Archaea. Ser201 is conserved in the HMG-CoA lyases of all organisms reported as well as Ser75, which is conserved in all HMG-CoA lyases save those of R. rubrum and P. mevalonii, in which it is replaced by an alanine.
Expression of Mutant Proteins in E. coli and Determination of Enzymatic ActivityThe expression system used to overexpress mature wild type and mutated HMG-CoA lyase was that described by Roberts et al. (36). E. coli JM105 cells were transformed with pTr-HL-wt, pTr-HL-75R, pTr-HL-201Y, and pTr-HL-204N plasmids or the empty vector (pTr-0), and three different cell clones for each plasmid construct were induced to express HL. Expression levels were the same in the three clones of each construct, and no expression was detected by Western blot in cells transfected with the empty vector (data not shown).
Specific HMG-CoA lyase activity from wild type and mutants was determined in the soluble fraction of crude extracts of transformed E. coli. Mutations S75R and S201Y completely abolished the enzyme activity, even when 75 µg of total protein extract was used in the assay. It was impossible to perform saturation kinetics and determine the Km value for these mutants. The calculated Vmax for the wild type was 9.5 units/mg protein and 1.35 units/mg for mutant D204N. The apparent Km of the expressed protein was 3.1 mM for the wild type and 6.6 mM for the mutant D204N. Therefore, the mutant decreased Vmax 7.1-fold and increased the Km 2.1-fold with respect to the wild type (Fig. 2). The catalytic efficiency (Vmax/Km) for the mutated enzyme was 6.6% of the wild type, which confirmed the aciduria observed in the patient.
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Structural Model of Human HMG-CoA LyaseA relationship was
found previously between the 3-hydroxy-3-methylglutaryl-CoA lyase
(HMGL)-like family of proteins
(22) and the TIM-barrel
hisA gene from Thermotoga maritima (Protein Data Bank entry
1qo2
[PDB]
(21), which suggested
that a ()8 barrel structure (also known as TIM barrel)
could be adopted by the HMGL-like family
(23). This type of structure
consists of an 8-fold repetition of a
-sheet-loop-
-helix-loop
motif, with the
-sheets forming the inner face of the barrel and the
-helices the external face. In addition, using methods based on
recursive intermediate sequence searching
(37) as well as the
hierarchical classification of proteins contained in the PROTOMAP data base
(38), a further relationship
was found between HMG-CoA lyase and the HisA (1qo2
[PDB]
) protein families. On the
basis of all these results, multiple alignment of both protein families was
performed (Fig. 3), and the
information extracted was used to construct a three-dimensional model of the
human HMG-CoA lyase, using the structure of T. maritima HisA protein
as a template (Fig. 4). This
model is a (
)8 barrel with short loops at the
NH2-terminal face, and long and probably non-structured loops at
the COOH-terminal face. The secondary structure prediction for the eight
-sheets and
-helices of the HMG-CoA lyase sequences calculated
using the PHD method (39,
40) is compatible with the
alignment made using sequence searching methodologies, showing a high
correlation between structural and sequence alignments.
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In the proposed model for human HMG-CoA lyase the side chains of the barrel
forming -sheets conform a central cavity, which is similar to the
structure of other TIM barrel-like enzymes such as methylmalonyl-CoA mutase
(PDB entry 7req
[PDB]
) (41). A
section of the proposed model (Fig.
4C, right panel) shows the contour of the
central cavity where the calculations from the macromolecular docking program
"Hex" localized the substrate, suggesting that
3-hydroxy-3-methylglutaryl-CoA crosses the cavity and locates the small
3-hydroxy-3-methylglutaryl head close to the His233-defined active
site. The location of the active enzyme locus in the COOH-terminal face of the
-barrel is consistent with the general disposition of the active sites
in the TIM barrel-like structures
(42).
Once the three-dimensional model for human HL was established, residues
affected by the three novel mutations described here (Ser75,
Ser201, and Asp204) and by all missense mutations
reported previously (Arg41, Asp42, Val70,
His233, Leu263, and Glu279)
(8,
9,
10,
13,
18) were localized in the
model. Interestingly, all the mutated residues (marked as red spheres
in Fig. 4) except
Glu279 are localized in the -sheets and form part of the
central cavity, where the HMG-CoA substrate is assumed to be accommodated.
When Val70, Ser75, and Ser201 are replaced by Leu70, Arg75, and Tyr201, the proposed substrate cavity is occluded by the longer side chain of the mutated amino acids, and the entrance of the substrate is prevented (Fig. 5), which is consistent with the complete absence of activity observed in expression studies performed with mutants S75R and S201Y.
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Some of the residues that can be mapped with confidence are those defined
previously as essential for the enzyme activity on the basis of mutational
studies. His233, reported as the main amino acid responsible for
HMG-CoA lyase catalysis, is located on -sheet 7 beside the substrate
cavity (Figs. 3 and
4B). Kinetic analysis
with site-directed mutants H233R and H233A showed a decrease in activity of 4
orders of magnitude (8),
without modification of the Km. In the model, the
catalytic His233 is located exactly at the site in which the
3-hydroxy-3-methylglutaryl group is connected with the CoA, thus validating
the model. His235 (suggested to have an important function on
cation binding) is located at 5.3 Å from His233, which could
explain the effect of site-directed mutants (H235A and H235D) on catalytic
activity (1:15 with respect to wild type) and substrate affinity
(Km was increased 4-fold)
(43).
Because Asp204 is close to His233 (less than 4 Å) in the model (Fig. 4B), we propose that the third novel mutation here described, D204N, could affect the activity of the enzyme by changing the polarity of the active center, which fits with the decreased Vmax observed in the expressed protein.
In addition, Arg41 and Asp42, which are on the first
-sheet (Fig. 3), are
close to the catalytic residue His233
(Fig. 4). Mutations in these
amino acid residues R41Q, D42H, and D42G, responsible for
3-hydroxy-3-methylglutaric aciduria in some patients
(13), produce changes of
charge, which may also affect the efficiency of catalysis. On the other hand,
Leu263 located close to
-sheet 8, surrounds the cavity and is
three amino acids away from Cys266, which is considered to be the
residue to which the acetoacetic group binds before leaving the enzyme after
catalysis (7). Mutation L263P
(18) may interfere with the
positioning of the acetoacetic group before leaving the enzyme. Mutation E279K
(9) is at the beginning of
-helix 8 (Fig. 3), and
the model cannot directly explain the absence of enzyme activity in this
mutated enzyme, because it is located outside the inner cavity
(Fig. 4). Because some proteins
that fold as TIM barrels utilize loop movements to close the active site once
the substrate is bound (44),
mutation E279K may block this effect in catalysis.
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DISCUSSION |
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Enzymes containing the ()8 barrel structural fold
account for up to 10% of all the enzymes of the central metabolism pathways
and support a wide variety of functions
(42). The amino acids
conforming the active locus are generally located at the COOH-terminal ends of
the
-sheets and at the loops that connect the
-sheets to the
-helices (42). Copley
and Bork (23), using the
Psi-Blast sequence-based search method
(24), provided compelling
evidence that most of the known and predicted TIM barrels found in central
metabolism share not only a structural fold but also a common evolutionary
origin. These authors also found related sequences in enzymes of central
metabolism whose three-dimensional structure has not yet been solved, although
it could be TIM barrel-like folds on the basis of sequence similarity. A
relation was found between members of the Pfam HMG-like
(22) family of proteins and
the TIM-barrel hisA gene from T. maritima (PDB entry 1qo2
[PDB]
(21)), leading to the
suggestion that a (
)8 barrel structure could be
adopted by the HMG-like family.
Two other independent approaches supported a direct sequence relationship between the human HMG-CoA lyase and the HisA (1qo2 [PDB] ) respective protein families. First, a program based on recursive intermediate sequence search methodology (37) confirmed a relation between those two families through a third family in the Pfam trp_syntA profile. In linking (or intermediate search) methods (4548) the two proteins are considered to be related if either their similarity score is above a certain cut-off or if there is a third protein related to both above a cut-off, following a transitive principle. Further evidence of sequence relation can be found at the PROTOMAP site (www.protomap.cs.huji.ac.il), which offers a hierarchical classification of the SwissProt proteins into clusters of related proteins (38). The relation described in PROTOMAP links at a lower reliability level "1e-0" the clusters 257 (HMGL proteins and related) and 503 (HisA proteins and related) through the small cluster 2889, related in addition to the cluster 163 (trp_synt proteins and related). On the basis of these sequence relations we suggest an alignment combining the Pfam HMGL_like and His_biosynth families of proteins (Fig. 3). In turn, this alignment was used to construct a three-dimensional model of the human HMG-CoA lyase, using the structure of the T. maritima HisA protein (PDB entry 1qo2 [PDB] ) as template.
The proposed structural model for HMG-CoA lyase corresponds to a
()8 barrel (Fig.
4) with short loops on the NH2-terminal face and, in
contrast, long and probably non-structured loops on the COOH-terminal face of
the
-barrel. The model is not accurate enough to infer the precise
location of all amino acids, because major movements of backbone and side
chains are expected (49).
However, it is sufficient to provide a suitable framework to determine the
general shape, the arrangement of the structural elements, and the
localization of key residues. In the case of T. maritima HisA protein
(PDB entry 1q02), the average in root mean square deviation, as deduced from
the data present in the FSSP (fold classification based on structure-structure
alignment of proteins) data base, is less than 3.5 Å for TIM barrels
with values of number of aligned residues and percent identity in the same
range as the ones between HMG-CoA lyase and HisA protein (227 residues and 9%,
respectively) (50).
Nine missense mutations producing 3-hydroxy-3-methylglutaric aciduria have
been described so far in the HMG-CoA lyase gene. Some of the residues that can
be mapped with confidence in the model have been defined previously as
essential to the enzyme activity based on mutational studies:
His233 (8,
18), which is considered as
the active site, Arg41, and Asp42
(13). In the present model,
all these amino acids, as well as Leu263
(18) and Asp204
(this paper), are located on the COOH-terminal face of the barrel forming
-sheets 1, 6, 7, and 8. All surround the end of a central cavity that
runs through the entire protein structure
(Fig. 4C), probably
forming part of an active locus, where the head of HMG-CoA fits to be split
off. The location of all these functionally essential residues in the
COOH-terminal face of the
-barrel is consistent with the general
arrangement of the active sites in the TIM barrel-like structures
(42). Because they are all
close to His233 in the modeled structure, these mutations could
affect the activity of the enzyme by changing the appropriate environmental
requirements of the active center. The kinetic behavior of D204N-expressed
protein is in accordance with the model; the catalytic efficiency is decreased
15.1-fold (6.6%) in respect to the wild type, whereas the substrate affinity
is barely modified (the apparent Km only doubles
in D204N in respect to the wild type). The loss of enzyme activity of the
mutants S75R and S201Y (this paper) and of V70L
(10) has an alternative
explanation within the proposed model. In the mutant proteins the side chains
of the Arg75 and of Tyr201 are much larger than those of
Ser75 and Ser201 (wild type) and may occlude the
proposed cavity, preventing the entrance of the substrate
(Fig. 5, A and
B). These changes may explain the lack of enzyme
activity.
These results indicated that in HL not only the amino acids that
participate directly in the catalytic reaction (Cys266 and
His233) but probably all the amino acids surrounding the substrate
cavity are critical for enzyme activity. Although Glu279 is outside
the central cavity (it is in -helix 8), it may play a putative role in
catalysis by utilizing loop movements to close the active site once the
substrate is bound, as has been shown in some enzymes that fold as TIM barrels
(44). This would correlate
with the finding that patient having mutation E279K did not develop lethal
symptoms (onset of the disease was at 7 months, residual HMG-CoA lyase
activity was 2% of the wild type, and patient development at 4.4 years is
normal) (9), and the
involvement of Glu279 in catalysis in this patient could be only
partially impeded by the E279K substitution.
Other possible speculations on the role of specific residues previously
identified as important for the enzyme activity, such as His235
(43) or Cys266
(7), can be based on their
location in the structural model. Thus, His235 is located at a
relatively short distance from the substrate (5.3 Å), which could
explain the effect of mutants H235A and H235D in both catalytic activity (1:15
with respect to wild type) and substrate affinity
(Km was increased 4-fold)
(50). Cys266, which
localizes in the COOH-terminal loop between -strand 8 and
-helix
8, could take part in enzyme function through putative loop movements, as
supposed for Glu279. Although these structural arrangements could
explain the lack of activity of the HMGL_HUMAN mutants described, these
structural data should be considered with caution, as they are derived from a
low resolution homology-based model.
Expression studies to identify the crystallographic structure of the HL protein are being carried out in our laboratory, in order to corroborate the model and improve the precision with which the amino acids are localized.
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FOOTNOTES |
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d Recipient of a fellowship from the Universitat Internacional de
Catalunya.
f Recipient of fellowship from the Diputación General de
Aragón.
l Present address: Dept. of General Pediatrics, University Children's
Hospital, Heinrich-Heine-University Düsseldorf, Moorenstrasse 5, D-40225
Düsseldorf, Germany.
m To whom correspondence should be addressed: Dept. of Biochemistry, School of Pharmacy, Avda. Diagonal 643, E-08028 Barcelona, Spain. Tel.: 34-93-402-4523; Fax: 34-93-402-4520; E-mail: hegardt{at}farmacia.far.ub.es.
1 The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HL,
HMG-CoA lyase; PDB, Protein Data Bank.
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ACKNOWLEDGMENTS |
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REFERENCES |
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