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Originally published In Press as doi:10.1074/jbc.M413281200 on May 12, 2005

J. Biol. Chem., Vol. 280, Issue 30, 27719-27727, July 29, 2005
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Characterization of Four Variant Forms of Human Propionyl-CoA Carboxylase Expressed in Escherichia coli*

Hua Jiang{ddagger}, K. Sudhindra Rao{ddagger}, Vivien C. Yee§, and Jan P. Kraus{ddagger}

From the {ddagger}Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado 80045 and the §Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, November 24, 2004 , and in revised form, May 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, {alpha} PCC and {beta} PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the {beta} subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in Km values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the kcat/Km ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47 °C, blue native-PAGE revealed a lower oligomeric form ({alpha}2{beta}2) in the three mutants not detectable in wild-type and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a {beta} PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Four different biotin-dependent carboxylases are known to play a central role in mammalian metabolic pathways, such as oxidation of odd-chain fatty acids, catabolism of branched amino acids, fatty acid synthesis, and gluconeogenesis. One of these is propionyl-CoA carboxylase (PCC,1 EC 6.4.1.3 [EC] ), which catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA in the mitochondrial matrix (1). This enzyme consists of two nonidentical subunits, {alpha} and {beta}, encoded by two different nuclear genes, designated PCCA and PCCB, respectively. Structural studies of the human enzyme have indicated that {alpha} PCC is 72 kDa in size, whereas {beta} PCC is 54 kDa. Overall, PCC has an {alpha}6{beta}6 structure (1, 2). Mutations in either gene result in an autosomal recessive disease, propionic acidemia (MIM 606054 [OMIM] ), which usually presents as a life-threatening ketoacidosis in the neonatal period with protein intolerance, vomiting, failure to thrive, lethargy, and profound metabolic acidosis symptoms. This disease can result in mental retardation and can be sufficiently severe to cause neonatal death (1).

The cDNAs for {alpha} and {beta} subunits have been cloned (accession numbers X14608 [GenBank] and X73424 [GenBank] , respectively) and the structure of the genes has been elucidated (3–7). With the sequence information available, it has been possible to identify mutations from propionic acidemia patients (8–10), and many new mutations have been found recently in different ethnic groups (11, 12). Currently, ~52 and 53 mutations have been reported in the PCCA and PCCB genes, respectively (for a continuously updated list of all reported PCC mutations see uchsc.edu/cbs/pcc/pccmain.htm). However, how these mutations affect the enzyme has not yet been established and how each mutation accounts for the patients' phenotype is difficult to determine because most of the propionic acidemia patients are compound heterozygotes. This poor correlation between genotype and phenotype presents a problem in understanding this disease. Our interest is in elucidating the structural and functional consequences of mutations.

Because PCC has been expressed in our laboratory in a chaperone-assisted bacterial expression system (13), several other studies have been carried out employing different expression systems and using crude cell extracts to address the effect of variations on stability and/or activity of the enzyme. A large number of PCCB mutations have been studied in the bacterial expression system (14–16), and in a SV40-transformed fibroblast expression system (17, 18). In addition, the effect of some PCCB mutations on the {alpha}-{beta} heteromeric and {beta}-{beta} homomeric subunit assembly have been studied by a mammalian two-hybrid expression system (19, 20).

In our previous expression study (15), we identified PCCB mutants (R165W, E168K, D178H, P228L, and R410W) that are PCC activity deficient in patient-derived fibroblasts but were found to be capable of expressing wild-type level PCC activity when assembled in our chaperone-assisted E. coli expression system. This result indicated that these mutations exert their pathogenic effect because of an inability to assemble correctly in patients' cells. In this paper, three mutations from this group, R165W, E168K, and R410W, have been chosen for verifying this assumption. The interspecies alignment of the {beta} PCC subunit from different organisms shows that the residues affected by the patient-derived mutations are completely conserved in all homologous proteins studied (18), suggesting their critical roles in proper protein function and structure (14, 15, 18). R410W, which was said to be in the putative propionyl-CoA binding site (21), appears to be the most common disease-causing mutation in Japan (3, 8). E168K, an allele prevalent in the Spanish and Latin American populations (6), was associated with a broad spectrum of clinical manifestation in patients, ranging from severe to mild in both the homozygous and compound heterozygous conditions.

Crystal structure of fully assembled PCC is not available as yet. Over 20 years ago, Haase and co-workers (2, 22) have suggested, based on electron micrographs of PCC from Mycobacterium smegmatis, that six spherical {alpha} subunits on the periphery are attached to a large central hexameric core ({beta}6). Recently, crystal structures of Propionibacterium shermanii transcarboxylase 12 S hexameric subunit (TC-12S) (23) and of Streptomyces coelicor {beta} PCC (scPCC) were reported (24). In both cases the {beta} subunits, in the absence of {alpha}-subunits, form homohexamers ({beta}6).

The R165W mutation is close to the position of E168K. The structural analysis with a {beta} PCC homology model indicated that Arg-165 and Glu-168 form a salt bridge with each other, and contribute to a local hydrogen-bonding network (23). A497V was found to result in some improved characteristics in enzymatic catalysis (15). It is relatively frequent among Spanish patients and was previously thought to be because of a single nucleotide polymorphism (6, 10, 15, 20). Here, we present results using our bacterial expression system from which we can obtain ample quantities of PCC enzyme by successfully purifying mutant forms of PCC to homogeneity to define biochemical and structural properties of the mutant proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Protogel acrylamide solution was acquired from National Diagnostics (Atlanta, GA). [14C]NaHCO3 was purchased from PerkinElmer Life Sciences (Boston, MA). Unless otherwise noted, all other chemicals were purchased from Sigma.

Purification of Recombinant Human PCC Enzymes from E. coli— Construction of the pPCCAB expression plasmid carrying both PCCA and PCCB sequences was described previously (13). Derivatives of this expression construct containing the desired mutations, the conditions for the growth of bacterial cultures, cell harvesting, and preparation of the cell lysate were described previously (14, 15). Cell debris and insoluble material were removed by centrifugation for 10 min at 5,000 x g at 4 °C and the supernatant fractions were further centrifuged for 35 min at 40,000 x g at 4 °C. The final supernatant thus generated was labeled crude extract.

Bacterial crude extracts were loaded onto a DEAE DE52 column (5 x 12.5 cm) (Whatman) previously equilibrated with 10 mM potassium phosphate, pH 7.0. The column was washed with 10 mM potassium phosphate, pH 6.5, followed by 30 mM potassium phosphate, pH 6.5. PCC was eluted with 70 mM potassium phosphate, pH 6.5. The eluate was concentrated on Amicon using XM50 membranes (Millipore) and then adjusted with 10x PBS buffer to a final concentration of 1x PBS solution. This solution was loaded onto a monomeric avidin column (2.5 x 10 cm) (Pierce) previously equilibrated with PBS, pH 7.4. Recombinant PCC was incubated on the affinity column for 30 min and then eluted with 0.5 mM biotin in PBS, pH 7.4, after washing with PBS, pH 7.4. The protein was concentrated on Amicon with XM50 membranes to ~1–2 ml, and loaded onto a HR SephacrylTM S-400 size-exclusion column (1.5 x 95 cm) (Amersham Biosciences). One main protein peak was collected. There was a small but much broader peak after this main peak in some of the variants containing primarily free {alpha} subunit. The purity of the preparations was ≥95% as judged by SDS-PAGE. The proteins were then stored at a final concentration of 5–10 mg/ml in 10 mM potassium phosphate, pH 6.5, or PBS, pH 7.4, at -80 °C.

Protein and Enzyme Assays—PCC activity was assayed as described previously with some modification (25). The reaction mixture contained 50 mM Tris-HCl, pH 8.0, 2 mM ATP, 125 mM KCl, 10 mM MgCl2, 3 mM propionyl-CoA, 0.5 mg/ml bovine serum albumin, PCC enzyme (~10 µg of crude extract or 0.1 µg of purified PCC), and 10 mM [14C]bicarbonate (specific activity 2 µCi/µmol) in a final volume of 50 µl, and incubated at 37 °C for 2 min. The reaction was terminated by adding 50 µl of 10% trichloroacetic acid. The mixture was centrifuged at 13,000 x g for 5 min and 50 µl of supernatant was dried in a scintillation vial in a heating block at 80 °C for 50 min. The dry residue was dissolved in 0.15 ml of H2O and 4 ml of OPTI-Fluor scintillation fluid (PerkinElmer Life Sciences) was added. The samples were counted in Beckman LS 3801 scintillation counter. A blank containing the assay mixture without propionyl-CoA was subtracted. Protein was determined by the Lowry method using bovine serum albumin as standard (26). One unit of PCC activity is defined as the fixation of 1 µmol of bicarbonate per min at 37 °C into trichloroacetic acid-soluble material.

Steady State Kinetics—Kinetic constants were determined in reaction mixtures containing 50 mM Tris, pH 8.0, 0.5 mg/ml bovine serum albumin, 125 mM KCl, 10 mM MgCl2, 2 mM ATP, and 10 mM with propionyl-CoA concentrations ranging from 0.1 to 9 mM. Reactions were run at 37 °C and reaction velocity was determined by the product amount produced at several time points. Kinetic constants and their standard errors were calculated by fitting the data to the Michaelis-Menten equation,

(Eq. 1)
using Origin 6.1 software (OriginLab Corp., Northampton, MA).

Protein Gel Electrophoresis and Western Blotting—Denatured proteins were separated by SDS-PAGE (27) using a 9% separating gel with a 4% stacking gel. Blue native-polyacrylamide gels (BN-PAGE) were prepared as described previously (28, 29) with some modification. Samples were loaded onto and run through a 5–13% gradient polyacrylamide gel with a 4% stacking gel. Electrophoresis was carried out at 4 °C for about 6–7 h. A high molecular weight calibration kit for electrophoresis (Amersham Biosciences) was used as the protein standard for BN-PAGE. The gels were stained with Simple Blue (Invitrogen, Carlsbad, CA). Western blotting transfer and detection conditions were as described previously (14). The PCC antiserum was affinity purified according to the method of Smith and Fisher (30).

Far UV Circular Dichroism—Circular dichroism spectra were determined with a Jasco J-810 spectropolarimeter (Jasco Inc.) at 4 °C using a fused quartz cuvette with 1-mm path length from 200 to 260 nm. Enzyme samples were at concentrations of 0.4–0.5 mg/ml in 10 mM potassium phosphate, pH 6.5. Data were collected at 0.2-nm increments. The results are expressed as the mean residue ellipticity, [{theta}]MRE (deg cm2 dmol-1),

(Eq. 2)
where [{theta}]obs is the observed ellipticity in millidegrees, [P] is the molar concentration of protein ({alpha}{beta} dimer), n is the number of residues per mol of protein molecule ({alpha}{beta} dimer), and l is the path length in centimeters. The percentages of {alpha}-helix and {beta}-sheet were calculated using a prediction program, K2D algorithm (31, 32).

Thermal denaturation experiments were carried out using a Peltier IR cell temperature controller and monitoring the CD changes at 222 nm with the changing temperature. The scans were conducted with protein at about 0.5 mg/ml in 10 mM potassium phosphate, pH 6.5, and the temperature was increased at a ramping rate of 1 °C/min from 20 to 90 °C. The data were analyzed according to Pace and Scholtz (33).

HPLC Analytical Size-exclusion Chromatography—HPLC analytical size-exclusion chromatography was performed on a BioSep-SEC-S 4000 size column (300 x 7.8 mm, Phenomenex) using a Beckman Gold HPLC system with the following mobile phase: PBS, pH 7.4, at a flow rate of 1 ml/min. The column was calibrated using the protein standards purchased from Bio-Rad.

Thermostability—Thermostability studies of the variant PCCs were carried out in two different ways. In the first case, enzymes (0.01 mg/ml) were incubated at 37 °C and small aliquots were removed at specific time intervals for activity assays. In the second case, enzymes (0.5 mg/ml) were incubated at three temperatures, 27, 37, and 47 °C for 30 min. At the end of the incubation, samples were taken for PCC activity assay and a 6.5-µg aliquot was applied to BN-PAGE. The gels were stained with Simple Blue (Invitrogen).

Molecular Modeling—Sequence alignments of human {beta} PCC, five Mycobacterium {beta} PCC from the NCBI data base, TC-12S, and scPCC show similar degrees of sequence conservation of ~50–70% over the entire polypeptide chain, strongly suggesting similar structures. Additionally, the individual segments that include the mutations are totally conserved among all four sequences. Specifically, these segments are in human {beta} PCC residues 163–169 GARIQEG, residues 407–410 GIIR, and 496–497 AA. Furthermore, the TC-12S crystal structure that was determined first was used to solve the scPCC crystal structure. A homology model of the PCC {beta}6 hexamer was constructed using the TC-12S crystal structure (Protein Data Base code 1ON3 [PDB] ) (23) as a template and the InsightII software package (MSI). The amino acid substitutions for the mutations and polymorphism were modeled using O (34) and molecular figures prepared using Molscript (35) and Raster3D (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of the Wild Type and Variant PCCs—The wild-type and variant forms of PCC were overexpressed using our previously described E. coli expression system (13) and were purified to near homogeneity as described under "Experimental Procedures" (Fig. 1). With the exception of the enzyme containing the A497V polymorphism, all of the PCC variants exhibited significantly reduced yield, typically 25% of that obtained for wild-type PCC (Table I). The relative activities of the variant enzymes compared with the wild-type greatly improved after purification. The A497V polymorphism demonstrated 30% higher specific activity than the wild-type enzyme, whereas the three mutants had comparatively lower specific activity, about 30–40% of wild-type PCC.


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  		TABLE I
Specific activity and yield of purified wild-type and mutant PCCs

 
Kinetic Properties of the Variant PCCs—The steady state kinetic constants of the variants compared with those of wild-type PCC are shown in Table II. We did not observe a significant difference in the Km values for propionyl-CoA among these enzymes. Although the A497V polymorphism had a higher Vmax value compared with the wild-type enzyme, the catalytic efficiency of this polymorphism was not changed based on the kcat/Km ratios. On the other hand, the Vmax and the catalytic efficiency of the three mutants were reduced to about 50% of wild-type PCC.


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  		TABLE II
Steady state kinetic constants of wild-type and mutant PCCs The values are expressed as a mean ± S.E., and each experiment was repeated 3–7 times.

 



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  		FIG. 1.
SDS-PAGE analysis of purified wild-type and mutant PCCs. Proteins, 5 µg each, were subjected to 9% SDS-PAGE.

 
Structural Studies of the Variant PCCs—The next step in our characterization of the variant enzymes was to look for changes in structure of these proteins. Initial structural studies were carried out by measurement of the far ultraviolet circular dichroism spectra, which are shown in Fig. 2. The spectra of the mutant variant PCCs were quite similar to that of wild-type. Based on statistical t tests, there were no significant differences in either {alpha} helical content or {beta} sheet content between the variants and wild-type, which were on average about 29 and 18%, respectively (Table III). These results suggest that the substitutions have no significant effect on the secondary structure of mutant PCC proteins.


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  		TABLE III
Apparent molecular mass (m), percentages of secondary structure content, and apparent denaturation temperature (Tm) of wild-type and mutant PCCs determined by HPLC exclusion chromatography and CD spectra, respectively The values are expressed as a mean ± S.E. of 3 repeats and 4–6 repeats for apparent molecular mass and secondary structure, respectively.

 
We utilized an HPLC size exclusion chromatography to study the quaternary structure of the PCC holoenzymes and the possible effect of the {beta} PCC substitutions on oligomeric assembly. With the purified forms of enzymes, variant and wild-type PCCs produced only one sharp protein peak and there was no significant difference in protein molecular mass between these variants and wild-type, which was about 800 kDa (Table III), consistent with the {alpha}6{beta}6 structure. This result was similar to our previous findings using a HR Sephacryl S-300 size exclusion column (15). Altogether, none of the substitutions caused any detectable structure changes on PCC oligomers.

Thermostability of the Variant PCCs—Another tool we employed to compare the properties of the wild-type and variant enzymes were thermostability studies using different approaches. The enzymes were first studied by incubating them at 37 °C for different time intervals followed by enzyme assays. By the end of the 40-min incubation period, the activities of the variants decreased to about 30% of the initial activity compared with 70% for the wild-type enzyme (Fig. 3). The thermal stability of the variant enzymes was also assessed by following changes in the far UV CD signal with increasing temperature. From the thermal denaturation curves (Fig. 4) and the data analyzed by a two-state model, it is clear that the wild-type enzyme undergoes a cooperative two-state transition between the native and denatured states with a Tm of 57.6 °C (Table III). The thermal denaturation profile of all PCC forms studied here are irreversible. The variant enzymes showed very similar Tm values when compared with wild-type enzyme (Table III). Thermal denaturation detected by the far UV CD signal reflects changes in the secondary structure, specifically {alpha} helix, of the monomeric protein species. Hence, comparable Tm values observed in this work clearly indicated that the overall thermal denaturation of PCC happened to be independent of the individual point substitutions. However, the denaturation curves of the variant enzymes did not overlay with that of wild-type enzyme, indicating formation of some intermediate species in the thermal unfolding. These intermediates could possibly reflect (a) the presence of higher order aggregates with the mutant enzymes just before initiation of thermal unfolding (40–55 °C) and (b) formation of some lower oligomeric forms at higher temperatures (58–60 °C) with the mutant enzymes. Further studies in this connection were not possible because of irreversibility of thermal denaturation and precipitation of protein.

Assembly and Stability of the Mutant PCCs—We performed native PAGE (25) to further confirm the molecular masses and assess the integrity of these polypeptides. The mutants and wild-type PCCs behaved very differently when the gel was running at room temperature instead of at 4 °C. Wild-type and A497V variant enzymes produced a distinct band, whereas the three mutants were smeared, suggesting that the quaternary structure of these mutants was somehow disturbed (data not shown). We wondered if there is a possibility that temperature affects not only the PCC activity of the mutant enzymes, but their oligomeric status as well. However, a standard native gel and an HPLC analytical size exclusion chromatography are not very useful to test this possibility because of the large size of PCC of about 800 kDa. PCC barely migrates even into 5% gel and the resolution on an HPLC exclusion column is not very high either. Thus, we employed BN-PAGE (28, 29) to examine the oligomeric status of the enzymes following their exposure to three different temperatures (27, 37, and 47 °C). Enzyme samples were analyzed by BN-PAGE after being incubated for 30 min and enzyme activity of each of the treated samples was assayed as well (Fig. 5). BN-PAGE is a "change shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. It provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, subunit composition, and degree of purity and for the detection of subcomplexes (29). Incubation at three temperatures did not result in any loss of activity of wild-type PCC, and the polymorphic A497V lost 40% of its activity only at the highest temperature of 47 °C. At the same time, the three mutants were much more sensitive to the increases in temperature. They lost about 30 and 85% of activities at 37 and 47 °C, respectively, consistent with our thermostability study mentioned above. BN-PAGE (Fig. 5) shows that wild-type PCC contains two main bands, very close to each other and migrating slower than the largest protein marker of 670 kDa. At the top of the gel, there are two weak bands, which are probably the aggregated forms of PCC. The polymorphic variant of PCC, the A497V, shows almost the same bands as the wild-type. The three mutant PCCs have one main band at the same position as the wild-type with a weaker band below. The bands at the top of the gel, which we assume to be aggregates, are darker than those in the wild-type lanes. There are no visible differences for each enzyme between treatments of 27 and 37 °C. However, following the treatment at 47 °C, the oligomeric status of each protein has undergone some changes. Wild-type and the polymorphism A497V show almost the same change, whereas the three mutants appear to belong to another group. In the wild-type and A497V lanes, the weaker bands below the main band became darker, especially in the A497V sample, and one or two new weak bands with a smaller molecular mass appeared. In contrast, the main band of the three mutants has lost a lot of its intensity and a new protein species with a molecular mass of ~330 kDa appeared. When the mutant proteins were treated at 52 °C, they became cloudy and the proteins were trapped in the stacking gel (data not shown).

We were interested what was the subunit composition of the new 330-kDa band. We isolated and analyzed this form of PCC by SDS-PAGE (Fig. 5, bottom right). The ~330-kDa species from each of three mutants dissociated into two polypeptides co-migrating with the {alpha} and {beta} subunits derived from the wild-type. The ratio of densities of the two bands was about 1:1.

We wondered whether the 330-kDa species might be already detectable in crude cell extracts of E. coli containing the plasmids expressing the variants. The crude cell extracts were analyzed by BN-PAGE followed by Western blotting with affinity-purified anti-PCC antibody. A crude cell extract derived from E. coli containing only the chaperone, GroES and GroEL plasmid, was used as a negative control and a purified PCC protein as a positive control. Again, the ~330-kDa band was observed in the crude extracts of the three mutants, whereas the wild-type and the A497V did not show it (Fig. 6). This finding means that very likely the same PCC oligomer is formed either in E. coli cells containing the mutant PCCs or by heating the purified human recombinant mutant protein.



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  		FIG. 2.
Circular dichroism spectra of wild-type and mutant PCCs. The protein concentrations were 0.5 mg/ml and the path length of the cell was 1 mm. Buffer for CD determination was 10 mM potassium phosphate, pH 6.5, and determinations were performed at 4 °C, 3–6 times for each enzyme.

 



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  		FIG. 3.
Thermal stability of wild-type and variant PCCs. The PCCs (0.01 mg/ml) were incubated in PBS, pH 7.4, and 4% glycerol at 37 °C. Samples were removed at the times indicated and enzyme activity was determined. Values shown represent the average of 4–7 independent experiments. In the interest of clarity, error bars are not shown on the graph but the S.E. was wihin 6% of the mean in all cases.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PCC consists of heterogeneous subunits in an equal ratio ({alpha}6{beta}6). Mutations have been found in both subunits (10). Prior studies have indicated that many mutations in PCCA result in the patient having little or no {alpha} PCC and {beta} PCC, and that the existence of {beta} subunit is dependent on the existence of {alpha} subunit (37, 38). The opposite, however, is not true. Mutations in PCCB may result in variable amounts of {beta} PCC or none at all, but {alpha} PCC remains stable in all cases.

In this paper, we chose three PCCB mutations in which immunoreactive protein was found, and that showed some residual enzyme activity in extracts of patient fibroblasts (15). When we expressed these mutations in E. coli and assayed their PCC activity in crude extracts, they were found to be capable of expressing wild-type level activity when assembled in our chaperone-assisted expression system (15). The three PCCB mutants (R165W, E168K, and R410W) demonstrated between 32 and 43% of wild-type enzyme activity after purification to homogeneity. In contrast, the polymorphic variant had nearly 140% of wild-type activity. Our goal was to elucidate the potential structural and functional effects of these substitutions on the mutant enzymes and to verify our assumption that these PCCB mutations may result in some impairment of assembly but little effect on catalytic activity.

Is the Catalytic Activity Affected by These Mutations?—We first investigated whether the lower activity of the mutants was because of a decreased affinity of the mutant PCCs for the substrate. Based on the study of interallelic complementation and sequence homology with the P. shermanii transcarboxylase 12 S subunit (TC12S), it was assumed that the propionyl-CoA binding site was located in the {beta} subunit and, furthermore, that mutation R410W was part of the propionyl-CoA binding site (21). Surprisingly, our steady state kinetic studies did not show any significant difference in Km values for propionyl-CoA among these variants and the wild-type enzyme (Table II), which indicated that these amino acids may not be involved in the propionyl-CoA binding site and that the Arg-410 residue was not responsible for propionyl-CoA binding, as presumed previously. The TC12S subunit whose crystal structure has been resolved recently (23) carries out a biotin-dependent carboxyl transfer reaction similar to that catalyzed by PCC and shares 40% sequence identity with the {beta} subunit of PCC (5, 23, 39). The TC12S-based homology model of the {beta} PCC subunit predicts a conserved protein fold, hexamer structure, and active site. The conserved PCC {beta} Arg-410 corresponds to TC12S Arg-387 and is located far from the MMCoA binding site, consistent with our kinetic study; the shortest distance between Arg-410 and MMCoA is 14 Å, through the body of the protein (Fig. 7) (23). Our purified mutant PCCs exhibited an approximate 50% reduction in catalytic efficiency (kcat/Km). Although this represents a significant impairment in PCC function, this decrease does not explain the low residual PCC activity found in patient-derived fibroblasts and the severe disease caused by the mutations studied here.



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  		FIG. 4.
Thermal denaturation of wild-type and variant PCCs monitored by CD changes at 220 nm. The protein concentrations were 0.5 mg/ml and the path length of the cell was 1 mm. Buffer for CD determination is 10 mM potassium phosphate, pH 6.5, and determinations were repeated 3 times.

 
Do These Mutations Affect the Secondary Structure, Protein Stability, or Dodecamer Assembly?—Previous work has indicated that the changes in the {alpha}/{beta} subunit ratio of PCC is the result of unsuccessful interaction of the {beta} subunit with the {alpha} subunit (38). In the previous expression studies, some PCCB mutations were shown to cause complete instability of the {beta} subunit, whereas other PCCB mutations, such as R165W and E168K, showed an increased {alpha}/{beta} subunit ratio compared with wild-type because of the presence of free {alpha} subunits (15). The purified mutant enzymes did not show any measurable differences in their secondary structures using the far UV CD approach (Fig. 2 and Table III). Size exclusion HPLC showed that the mutant PCCs had a molecular mass around 800 kDa, consistent with the {alpha}6{beta}6 oligomeric structure. Furthermore, blue native PAGE further confirmed the molecular masses and assessed the integrity of these polypeptides (Fig. 5, bottom). These data suggested that all the enzymes had similar quaternary structures. We noticed, however, that the activity of the mutant enzymes decreased significantly following incubation at 37 or 47 °C (see Figs. 3 and 5, top). In addition to the changes in activity, the oligomeric status of these mutants changed when the enzymes were exposed to increased temperatures (Fig. 5, bottom). These results implied a reduced stability of the assembled mutant enzymes, and the reduced stability was likely because of weak or improper interactions between the mutant {beta} PCC subunits or between the {alpha} PCC and mutant {beta} PCC compared with wild-type subunits.



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  		FIG. 5.
The change of activity and oligomeric status of wild-type and variant PCCs following incubations at different temperatures. The enzymes were incubated at different temperatures as described under "Experimental Procedures." The activity assay was carried out immediately after the incubation (top) and an aliquot was analyzed on a BN-PAGE gel (bottom). Each treatment was repeated 4 times and the error bars indicate the S.E. Lanes a, b, and c for each sample show the enzymes after incubation at 27, 37, and 47 °C for 30 min, respectively. M stands for the molecular mass marker. To determine the composition of the new ~330-kDa band that has appeared in the mutant lanes after incubation at 47 °C, the gel piece containing this band was cut out, boiled in SDS gel sample buffer for 5 min, and inserted into a well of a 9% SDS gel. First and second lanes (bottom right) show the analysis of the 330-kDa band and wild-type enzyme, respectively.

 



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  		FIG. 6.
BN-PAGE and Western blot analysis of the oligomeric status of wild-type and variant PCCs in the crude extract of E. coli. The proteins, 50 µg, were subjected to BN-PAGE and PCC was detected after blotting with anti-PCC antibody. Lanes 1–7 are wild-type, A497V, R165W, E168K, R410W, GroESL, and purified PCC, respectively. Lane M is the molecular mass marker.

 
In addition, a new oligomeric form of PCC of about 330 kDa appeared after the mutant PCCs had been exposed to increased temperatures (Fig. 5, bottom). Moreover, the same 330-kDa form was detected in the corresponding crude E. coli extracts (Fig. 6). Based on a subsequent SDS-PAGE analysis (Fig. 5, bottom, first lane), this oligomeric form appears to be a tetramer of {alpha}2{beta}2 structure. It is not unreasonable to suggest that PCC assembly sequence involves formation of tetramers, {alpha}2{beta}2, and that the enzyme is, in fact, a trimer of tetramers. This is similar to the homologous TC12S hexamer, which dissociates into dimers, and thus is a trimer of dimers (40–42). Thus, the PCCB mutations likely weaken the interactions in the context of the {alpha}6{beta}6 dodecamer rather than in an isolated {beta}6 hexamer.

We did not observe significant differences in secondary and quaternary structure detected by CD and HPLC because of the following reasons. First, the enzymes we tested have been purified and the unassembled proteins have been removed. The increased {alpha}/{beta} subunit ratio (15) and the small PCC form observed in crude cell extracts of the mutants (Fig. 6) indicated that smaller cross-reactive forms existed and that they were the result of the mutations. Second, the resolution of the HPLC column was not high enough to separate the different oligomeric PCC forms as efficiently as the BN-PAGE because of the extremely high molecular mass of PCC. Based on the BN-PAGE results and our Western blotting experiments (Fig. 5 and Fig. 6), it appears that these mutations result in some misfolding and/or the production of unassembled protein during synthesis of recombinant mutant PCC. Even though some of the mutant proteins are capable of assembly into dodecamers, their near inactivation at 47 °C and disassociation to lower oligomeric form ({alpha}2{beta}2) indicate that they are significantly reduced in terms of stability compared to the wild type enzyme. The lower specific activity of the mutants and the lower Vmax observed in kinetic studies compared with wild-type may be the side effect of the dodecamer instability rather than because of any impairment of catalytic ability.



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  		FIG. 7.
Homology model of {beta} PCC. Two orthogonal views of the {beta}6 hexamer predicted to form part of the {alpha}6{beta}6 dodecamer. Monomers are shown as coil structures, with one dimer pair colored purple and cyan. The six MMCoA substrates (yellow ball-and-stick, as observed in the homologous TC-12S crystal structure) and the six biotin molecules (red ball-and-stick, as observed in the homologous scPCC crystal structure) are bound between monomers at the dimer interface. Mutation and polymorphism sites are shown for the purple and cyan dimer pair. R165W, (blue spheres), E168K (green spheres), and R410W (red spheres) are located near the dimer interface, with the former two adjacent to the substrate binding site, whereas A497V (yellow sphere) is located near an inter-dimer (intra-trimer) interface.

 
{beta} PCC Homology Modeling—Analysis of the {beta} PCC homology model provides explanations for the possible effects of the variants on quaternary structure. Arg-165, Glu-168, Arg-410, and Ala-497 are all conserved in Mycobacterium {beta} PCC, TC12S, and scPCC bolstering use of the homology model for structural interpretations. Significance of {beta}6 is further provided by the electron micrograph of M. smegmatis PCC (2) that suggests a central core of {beta}6 surrounded by six {alpha} subunits. There is no evidence of formation of {beta}6 in our system where both types of subunits are expressed in stoichiometric amounts. The mutants and the polymorphism alter residues, which are located near the surface of the monomeric protein fold, and none are predicted to cause global protein misfolding or instability. Instead, the three mutants are likely to have local conformational differences relative to wild-type, whereas the polymorphism is predicted to cause little or no local conformational change. For example, within the same {beta} monomer, Arg-165 and Glu-168 form a salt bridge with each other, and contribute to a local hydrogen-bonding network involving Asp-105 and Cys-90, which serves to tether two surface loops and appears to be important for the local structure (Figs. 7 and 8). It should be mentioned that Asp-105 is a totally conserved residue among all four sequences. The loop containing Arg-165 and Glu-168 is also adjacent to the opposing {beta} monomer in the dimer pair. Because the R165W and E168K mutants are predicted to disrupt the local hydrogen-bonding network, the absence of these interactions is likely to result in a local conformational change. This, in turn, could alter the intermolecular interface in the {beta} dimer pair, and consequently hexamer/dodecamer formation and/or stability.

R410W is expected to have a similar effect. Arg-410 is also located at the {beta} dimer interface, albeit distant from the active site: it hydrogen bonds across the dimer interface with Ser-254 in the opposing {beta} monomer. It should be mentioned that Ser-254 is a totally conserved residue among all four sequences. The R410W mutant introduces sterically unfavorable short contacts that require a change in local main chain conformation for removal. This local structural change at the {beta} dimer interface would likely also alter hexamer/dodecamer formation and/or stability.

However, in the absence of the crystal structure of the {alpha}6{beta}6 dodecamer of PCC per se, the arguments above provide the best clue as to how these mutations could affect the enzyme. The sequence of events in the overall assembly of {alpha}6{beta}6 PCC from individual {alpha} and {beta} peptide syntheses remains an open question although there is evidence for {alpha}2{beta}2 tetramer in the degradation process and thus the {alpha}2{beta}2 tetrameric species may be involved in the overall assembly of {alpha}6{beta}6 PCC.

Some Mutant PCCs Respond Strongly to Molecular Chaperones—It was reported that by using the mammalian two-hybrid system, mutations R165W and E168K resulted in a 40% reduction in {alpha}-{beta} and {beta}-{beta} subunit assembly. Lowering the temperature from 37 to 27 °C, however, allowed the rescue of assembly of both mutants to normal levels (19, 20). Temperatures lower than the physiological one have a positive effect on protein folding, mimicking the role displayed by molecular chaperones (43, 44). In our expression system, PCC is expressed in the presence of the bacterial chaperone proteins, GroEL and GroES (13), which are homologous to the eukaryotic chaperones, hsp60 and hsp10. Chaperone proteins assist polypeptide folding by unfolding nonfunctional conformations, followed by reinitiating the folding process to escape protein degradation (45). Co-expression of human polypeptides with bacterial chaperone proteins has been shown to correct the misfolding of some mutant forms of phenylalanine hydroxylase (46). Misfolded proteins are typically destined for proteolytic degradation (20). Missense mutations not situated in the active site or cofactor binding site may have an effect on protein stability by producing nonfunctional conformations that lead to degradation or aggregation (44). The levels of chaperone proteins in our expression system do not mimic those levels found in eukaryotic cells because we have overexpressed GroES and GroEL. Consequently, the residual activity of our purified proteins is higher than that seen in vivo. Therefore, we suggest that these mutant PCCs can be folded into a conformation and can be assembled into a holoenzyme, which restores some of their wild type activity with the help of chaperone proteins. However, these mutant polypeptides still maintain some degree of misfolding, resulting in decreased stability and enzymatic activity. Further tests in vivo using pulse-chase experiments may help elucidate the level of mutant protein production in patient cells and the turnover rate of these subunits. How the chaperones affect the folding and/or assembly of PCC and whether molecular or chemical chaperones can rescue these mutant proteins in vivo are the subject of ongoing studies and these approaches may provide a way to help patients who suffer from propionic acidemia because of these or similar mutations.



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  		FIG. 8.
R165W and E168K mutants in the {beta} PCC homology model. Left, Arg-165 and Glu-168 in wild-type {beta} PCC participate in a hydrogen-bonding network (dashed lines), which includes Cys-90 and Asp-105. These interactions serve to tether two surface loops (thick purple coil). The loop containing Arg-165 and Glu-168 is adjacent to the opposing monomer (cyan) in the dimer pair, and flanks the binding site for the MMCoA substrate (thin ball-and-stick, from the TC-12S crystal structure); the biotin site (identified in the scPCC crystal structure) is further away. Right, R165W and E168K (green bonds and carbon atoms) in {beta} PCC mutants are predicted to disrupt the local hydrogen-bonding network.

 
The variant A497V has always been seen in cis together with either mutation R165Q or V217K/218del2 on Spanish mutant alleles (18, 20). We were unsure how this variant contributed to the pathogenesis of propionic acidemia. Purification of A497V, followed by thermostability studies, indicated that the A497V-containing enzyme was less stable than wild-type PCC. On the other hand, this substitution had no effect on substrate binding, catalytic efficiency, or oligomeric assembly. In contrast to the three mutations, Ala-497 is located at the trimer interface, i.e. between dimer pairs, and although it is a conserved residue, it is in a less conserved local environment. Compared with the mutations studied here, the larger side chain of the A497V variant introduces short contacts, which are expected to be easily accommodated by little or no change in local protein conformation, and thus little or no change in quaternary structure/stability. Our experiments point to some small local conformational changes but no significant impact on the oligomeric status and support the notion that A497V is a single nucleotide polymorphism as concluded from previous work on interactions between {alpha}-{beta} or {beta}-{beta} subunits (19, 20) or from PCC activity measurements (15). The potential modifying effect of A497V on R165Q has been investigated in the crude extract of SV40-transformed fibroblasts, and no measurable effects were seen (18).

Genotype-Phenotype Correlations—Genotype-phenotype inconsistencies exist for some of these mutations. It is unclear why patients with the same allelic mutations have different clinical symptoms. It has been speculated that environmental and genetic factors (such as gene modifiers or sequence alterations in other chaperonin genes that mildly impair folding) may combine to modulate the outcome of clinical disease (44). Although we have not investigated sequence polymorphisms among the chaperone proteins in these patients, the increased enzymatic activity seen in our bacterial expression system suggests that an overabundance of chaperone proteins may produce significantly higher enzymatic activities. These higher activities, in turn, may lead to milder clinical symptoms. The phenotypic variability between patients, therefore, may be a consequence of PCC mutations in addition to polymorphisms in chaperone proteins.


   FOOTNOTES
 
* This work was supported by National Institutes of Health NICHD Grant P01HD08315 (to J. P. K.) and National Science Foundation MCB Grant 0401654 (to V. C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Pediatrics, University of Colorado School of Medicine, Mail Stop 8313, P. O. Box 6511, Aurora, CO 80045-0511. Tel.: 303-724-3812; Fax: 303-724-3838; E-mail: jan.kraus{at}uchsc.edu.

1 The abbreviations used are: PCC, propionyl-CoA carboxylase; BN, blue native; PBS, phosphate-buffered saline; scPCC, Streptomyces coelicor PCC; MMCoA, methylmalonyl-CoA; TC12S, transcarboxylase 12 S subunit; HPLC, high performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We appreciate the generous help of Dr. Todd Kelson and Nina Frank, M.S. in critical reading and editing this manuscript. We also thank Drs. Frank Frerman for constructive criticism and Paul Cachia for help with CD measurements.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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