Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD) K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein Linda P. O’Reilly 1, *, Brage S. Andresen 2 and Paul C. Engel 1 1 Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland 2 Research Unit for Molecular Medicine, University Hospital, Skejby Sygehus, Aarhus, and Institute of Human Genetics, Aarhus University, Denmark Keywords active site; enzyme deficiency; medium chain acyl-CoA dehydrogenase (MCAD); point mutations; protein folding Correspondence P. C. Engel, Department of Biochemistry, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Fax: +353 12837211 Tel: +353 17166764 E-mail: Paul.Engel@ucd.ie Website: http://www.ucd.ie/biochem/ Enzymes Medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3); long chain acyl-CoA dehydrogenase (LCAD; EC 1.3.99.13); short chain acyl-CoA dehydrogenase (SCAD; EC 1.3.99.2); glutaryl-CoA dehydrogenase (GCD; EC 1.3.99.7); isovaleryl-CoA dehydro- genase (IVD; EC 1.3.99.10); electron trans- ferring protein (ETF; EC 1.5.5.1). *Current address Department of Molecular Genetics and Biochemistry, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15261, USA (Received 13 January 2005, revised 24 June 2005, accepted 25 July 2005) doi:10.1111/j.1742-4658.2005.04878.x Two novel rare mutations, MCAD842GfiC (R256T) and MCAD 1166AfiG (K364R), have been investigated to assess how far the bio- chemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the syn- thesized mutant protein only exhibited activity when the gene for chapero- nin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chapero- nin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moder- ate kinetic impairment, whereas the R256T protein was again totally inac- tive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermo- stable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed. Abbreviations ACAD, acyl-CoA dehydrogenase; BCIP, 5-bromo-4-chloro indol-3-yl phosphate; DCPIP, 2,6-dichlorophenolindophenol; ETF, electron- transferring flavoprotein; GCD, glutaryl-CoA dehydrogenase; INT, 2-(4-iodophenyl) 3-(4-nitrophenyl) 5-phenyl-tetrazolium chloride; IVD, isovaleryl-CoA dehydrogenase; NBT, 2,2¢-di-p-nitrophenyl 5,5¢-diphenyl 3,3¢-(3,3¢-dimethoxy-4,4¢-diphenylene) ditetrazolium chloride; PES, phenazine ethosulphate; SCAD, short-chain acyl-CoA dehydrogenase; VLCAD, very long chain acyl-CoA dehydrogenase. FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS 4549 Medium chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein that catalyses one of the recurrent steps in the b-oxidation of fatty acids. MCAD functions by removing two reducing equiva- lents from the fatty acyl substrate, and donating them to electron-transferring flavoprotein (ETF), which ulti- mately feeds them to the electron transport chain to generate ATP [1]. Within the active site, the substrate fatty acyl chain is sandwiched between the isoalloxa- zine ring of the FAD prosthetic group and the carb- oxyl group of the catalytic glutamic acid residue (E376). This residue removes one hydrogen as a proton from the C-2 position of the fatty acid thioester. The other is simultaneously removed as a hydride ion by the N-5 position of the isoalloxazine ring. With this oxidation step the FAD becomes reduced to FADH 2 [2]. The reducing equivalents are then transferred from the reduced flavin of MCAD to ETF, and the enoyl- CoA is released to be further degraded via the b-oxida- tion cycle [1]. Defects in MCAD accordingly impair the ability to degrade fatty acids, and MCAD deficiency is the most frequently diagnosed clinical defect of fatty acid meta- bolism [4], with a frequency of 1 : 15 000 in the USA population [5]. Eighty per cent of patients are homo- zygous for the common K304E (MCAD985AfiG) mutation, and a further 18% have this mutation in one of the two defective alleles [5]. Symptoms of MCAD deficiency cover a broad spectrum, ranging from hypoglycaemia to seizures, coma and sudden death, and usually present at a time of metabolic decompensation associated with fasting or viral illness [4]. However, some individuals carrying the genetic defect remain asymptomatic throughout life [6–8]. A variety of rare MCAD mutations have been identified through screening programmes, and, where they have been characterized at the protein level, the defect has thus far mainly been in folding [9,10]. It is not yet clear whether this merely reflects a greater statistical likelihood of impaired folding than of impaired catalysis or cofactor binding. Here we describe two novel rare mutations with contrast- ing enzymological consequences. The first, R256T (MCAD842GfiC), was found as a compound heterozygote with K304E in a screening programme in the USA. This newborn exhibited elevated hexa- noyl, octanoyl and decenoyl carnitine levels, indica- ting MCAD deficiency. This mutation has previously been reported in four siblings, who, though exhibiting a biochemical profile indicative of MCAD deficiency (i.e. elevated hexanoylglycine in urine), have so far remained clinically and developmentally normal [11]. The second mutation, K364R (MCAD1165AfiG), was detected in homozygous form in a UK child of Asiatic origin exhibiting biochemical indications of MCAD deficiency. In order to assess whether these newly discovered missense mutations affect the ability of the MCAD protein to fold, expression levels in Escherichia coli were monitored, with and without the co-overexpres- sion of GroEL and GroES [6,9,12]. Stability of the mutant proteins in this model system was further investigated by determining the effect of temperature on the enzyme activity and structure. The MCAD mutants were also purified so that the kinetic parame- ters and stability of the homogeneous proteins could be investigated. Interaction with the natural electron acceptor, electron-transferring flavoprotein (ETF) was also tested, to give a more complete picture of the bio- chemical outcome of these point mutations. Results The effect of chaperonin on the ability of the mutant proteins to fold For a number of other MCAD point mutations, co-overexpression of the GroELS genes has been shown to rescue enzyme activity, suggesting that these mutations may affect folding in vivo [6,12]. Therefore the GroELS genes were overexpressed with those for each of the MCAD mutants, K364R and R256T, and for the wild-type enzyme, in E. coli cells grown both at 31 °C and 37 °C, and the effect on activity (ferri- cenium assay), and tetramer assembly (as determined by western blot analysis of native gels) was investi- gated (Fig. 1). At 31 °C, wild-type MCAD gave the highest level of activity of the three proteins, with 9550 nmol ferriceniumÆmg )1 Æh )1 (Fig. 1A). This increased to 11 700 nmol ferriceniumÆmg )1 Æh )1 in the presence of chaperonin, the moderate extent of this change pre- sumably reflecting successful unassisted folding for most of the MCAD. In the absence of chaperonin, increasing the growth temperature to 37 °C decreased the activity of wild-type MCAD by 55%, to 5340 nmol ferriceniumÆmg )1 Æh )1 . Co-expression of the chaperonin genes increased the MCAD activity nearly fourfold to 19 100 nmol ferriceniumÆmg )1 Æh )1 , an increase of almost 60% when compared with growth at 31 °C. This figure may in part reflect upregulation of both MCAD and chaperonin. It is clear, however, that, under these more stressful (though physiological) con- ditions of temperature, even the wild-type enzyme depends upon chaperonin for optimal folding in this recombinant overexpression system. MCAD catalysis (R256T) and folding (K364R) mutants L. P. O’Reilly et al. 4550 FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS No MCAD activity was detected in lysates of cells expressing R256T, but, at both temperatures, the fold- ing of this mutant protein appears to be even more successful than for wild-type enzyme, as judged both by the levels of MCAD tetramer present on the west- ern blot and by the minimal effect of chaperonin (Fig. 1B). This suggests that the R256T mutation harms the function of the folded protein rather than the ability of the protein to fold. K364R, by contrast, appears to be a severe tempera- ture-sensitive folding mutation. In the absence of chap- eronin, no activity was detected at either growth temperature (Fig. 1A). With chaperonin, the activity expressed at 31 °C (2950 nmol ferriceniumÆmg )1 Æh )1 ) could be rescued to 25% of the wild-type figure, but on increasing the growth temperature this level dropped to 6% (751 nmol ferriceniumÆmg )1 Æh )1 ) (Fig. 1A). These diminished activities, as compared with wild-type MCAD, clearly also correlate with decreased amounts of MCAD protein detected in the western blots (Fig. 1B). Comparison of activity levels of the mutant proteins, as determined by activity staining The apparent inactivity of R256T was further investi- gated to determine whether this was specific to the fer- ricenium assay, or whether this mutation renders the enzyme entirely catalytically inactive. Extracts of cells cultured at 31 °C were run on native PAGE. The sen- sitive activity stain, utilising phenazine ethosulphate (PES) and p-iodonitrotetrazolium violet (INT) as elec- tron acceptors, was used, and the gel was western blot- ted for direct comparison of the amount of folded tetramer with the activity level (Fig. 2). In general, the activity staining correlated well with the corresponding ferricenium assay results (Fig. 1A) and also compared well with the amount of folded tetramer present, for both wild type and K364R (Fig. 2A). As the stain is so sensitive, a slight staining could be seen for K364R in the absence of chaperonin, even though the ferri- cenium assay detected no activity. However, even with the increased sensitivity, R256T gave no indication of activity. Kinetic parameters of the purified variant proteins The mutant and wild-type proteins were purified to homogeneity by anion exchange and dye affinity chro- matography, with a novel substrate elution procedure proving very effective in securing a pure protein prod- uct. The results of kinetic analysis are displayed as Michaelis–Menten plots in Fig. 3A. The K m , V max (as determined by the Direct Linear and Wilkinson meth- ods), and k cat values are shown in Table 1. The K m value of wild-type MCAD for octanoyl-CoA, 3.69 lm, compares well with the published value of 3.4 lm [13,14]. Although the K m is increased somewhat for the K364R mutant protein (5.53 lm), the k cat is not significantly decreased. R256T was also purified, but, even at a final concentration of 21.4 lgÆmL )1 in the assay, this mutant protein still showed no catalytic activity. The activity levels were also determined using the natural electron acceptor, electron-transferring flavo- protein (ETF) (Fig. 3B), and, as expected, were very A B Fig. 2. Comparison of (A) western blot (showing the amount of tetramer formed) and (B) activity stain (showing the activity level of tetramer), of wild type (WT), R256T, and K364R MCAD both in the presence (+) and absence (–) of co-overexpressed chaperonin GroE in E. coli cells, grown at 31 °C. A B Fig. 1. Comparison of wild type (WT), R256T, and K364R MCAD both in the presence (+) and absence (–) of co-overexpressed chap- eronin (GroE), in E. coli, grown at 37 and 31 °C. (A) This shows the activity levels as determined by ferricenium activity assay (error bars ¼ standard deviation of the mean result). (B) Western blot analysis of native PAGE gels, indicative of relative amounts of sol- uble tetramer. L. P. O’Reilly et al. MCAD catalysis (R256T) and folding (K364R) mutants FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS 4551 low in comparison with activities measured with the ferricenium acceptor. Again, even with the natural acceptor, R256T showed no catalytic activity. K364R showed a marginal decrease in activity to a level about 9% lower than wild type, suggesting that the primary effect of this mutation is not on the catalytic activity of the enzyme. Absorption spectra of the purified mutant proteins To assess whether these mutations affected the affinity of the enzyme for the bound prosthetic group, FAD, absorption spectra were obtained. As FAD absorbs strongly at 450 nm, the ratio A 450 ⁄A 280 gives a rough indication of the amount of FAD bound to the puri- fied protein (assuming no major change in the extinc- tion coefficient of the bound cofactor in the case of the mutants) [15]. The peak absorbance ratios showed first of all that the wild-type enzyme as purified here (ratio ¼ 7.2) is somewhat depleted of FAD because the value should be 5.2–5.5. This probably reflects the rigorous procedure to remove excess FAD added dur- ing the purification, and certainly does not indicate the level of contamination by other protein, to judge from SDS ⁄ PAGE. The ratios for R256T (8.8) and K364R (9.6), both handled in exactly the same way as the wild-type enzyme, suggest that both may be slightly weakened in their affinity for FAD, with K364R the most affected. As this is an unstable protein, the FAD binding may be less secure than with a more tightly folded protein. The catalytic competence of the enzyme is in any case not greatly affected, suggesting that any FAD binding impairment cannot be the primary dele- terious effect of this mutation. As the reduction in affinity is not as great for R256T, FAD depletion is clearly not responsible for the inactivity of the R256T mutant protein. Thermostability of the purified mutant proteins The effect of temperature on MCAD stability was directly investigated by incubating aliquots of each purified protein (10 lgÆmL )1 ) at various temperatures (4–55 °C) for 10 min. Each aliquot was also subject to western blot analysis, so that the activity could be compared with the amount of native tetramer present. The thermostability curve (Fig. 4A) shows that K364R is less stable than wild-type, with 50% residual activity after 10 min at approximately 48 °C, compared with 58 °C for wild-type. At 55 °C K364R was completely inactive after 10 min. R256T remained inactive at all temperatures. The western blots compared well with A B Fig. 3. (A) Michaelis–Menten plot showing the substrate kinetics of the purified mutant protein K364R and wild-type MCAD (WT). (B) Comparison of the activity levels of purified K364R and wild- type MCAD, as determined by both the ferricenium and ETF assays (error bars indicate standard deviation of the mean result). Table 1. K m (lM), V max (nmol ferriceniumÆmg )1 Æmin )1 ) (as deter- mined by Direct Linear and Wilkinson methods), and k cat (s )1 ) for octanoyl-CoA oxidation. K m V max k cat Wild-type MCAD Direct linear 3.69 24.4 · 10 3 19 Confidence limits (68%) 3.23–4.03 23.8–24.9 · 10 3 Wilkinson 3.68 24.6 · 10 3 19.1 Standard error 0.305 621 K364R Direct linear 5.38 25.5 · 10 3 19.8 Confidence limits (68%) 5.09–5.87 25.4–25.6 · 10 3 Wilkinson 5.67 25.6 · 10 3 19.9 Standard error 0.186 274 MCAD catalysis (R256T) and folding (K364R) mutants L. P. O’Reilly et al. 4552 FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS the enzyme activity results (Fig. 4B). Wild-type MCAD shows a large amount of tetramer still present at 55 °C, at which temperature 68% enzyme activity remains. For K364R there was a dramatic decrease in the tetramer level at 50 °C, corresponding to the loss of enzyme activity. The R256T mutant protein, although not as thermostable as wild-type, shows con- siderably more structural stability than the K364R mutant, with a higher amount of tetramer remaining at 50 °C. Sequence alignment and position of the mutations in the three-dimensional structure The acyl-CoA dehydrogenase (ACAD) family shares 30–35% sequence homology within a species, and each individual ACAD member shows 85–90% sequence identity between mammalian species [16]. The align- ment (not shown) of the human MCAD sequence with published MCAD and short-chain acyl-CoA dehydro- genase (SCAD) sequences from various other sources shows that R256 is completely conserved across all the species studied, indicating the importance of this resi- due. The three-dimensional structure of porcine MCAD, which differs in sequence from the human enzyme at fewer than 10% of the positions [17,18], has been solved to 2.4 A ˚ (Fig. 5) [17]. In this structure, R256 is in very close proximity to the catalytic residue E376. During catalysis the E376 sidechain swings towards the Ca atom of the substrate, in order to abstract the proton [17]. It seems likely that the fixed charge of the guanidino group of R256 stabilizes the catalytic carboxylate in the correct position for cata- lysis. It is thus not surprising that removal of the con- served positive charge in the mutant R256T prevents catalysis. Indeed this residue has recently been studied in rat MCAD, where the arginine was mutated to alanine, lysine, glutamine and glutamic acid. The authors found that the lysine mutant exhibited signifi- cantly reduced activity, whereas the other variants were completely inactive [19]. As expected from the extensive sequence homology, the recently solved structure of a mammalian SCAD shows a high degree of similarity to MCAD [20]. At the amino acid position in SCAD corresponding to K364 in MCAD there is an arginine residue, R356 in the SCAD sequence. It is striking that this conserva- tive substitution is identical to the clinical mutation in the present case. Interestingly, though, this position in SCAD has also previously been identified as the site of a clinical mutation R356W [20]. This was found in a female newborn, who presented with hypotonia, seizures and developmental delay [21]. Another highly homologous ACAD, glutaryl-CoA dehydrogenase Fig. 4. Thermostability of purified wild-type (WT) MCAD and K364R mutant protein (10 lgÆmL )1 ) over increasing temperature. (A) This shows the thermostability as determined by the ferricenium assay after a 10-min incubation at the temperature indicated (error bars ¼ standard deviation of the mean), and (B) the amount of tetramer present, as determined by western blot analysis of native PAGE gels. Fig. 5. Structure of pig MCAD, solved with bound octanoyl CoA [17], is modelled using NCBIs Cn3D programme (PDB: 3MDE) [38]. The catalytic residue at position 376 and R256 are highlighted in yellow. Also shown is the octanoyl CoA (8-CoA) and FAD. L. P. O’Reilly et al. MCAD catalysis (R256T) and folding (K364R) mutants FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS 4553 (GCD), also has a frequent mutation at the analogous position (R402W), which exhibited only 3% activity when compared with wild-type in E. coli [22]. This position is also the site of a missense mutation in iso- valeryl-CoA dehydrogenase (IVD) (R363C), showing both reduced stability and activity. The activity was undetectable with the PMS ⁄ DCPIP assay, and only 0.01 lmol ETFÆ min )1 Æ(mgÆprotein) )1 when measured using the highly sensitive ETF fluorescence quenching assay [16,23]. In very long chain acyl-CoA dehydro- genase (VLCAD), an analogous mutation (R410H) has also been found to cause disease in three compound heterozygote patients [24,25]. To the authors’ know- ledge, this residue is the most frequently mutated posi- tion across the entire ACAD family. Modeling of this residue, using the rat SCAD crystal structure, sugges- ted disruption of the local bonding and steric hin- drance by the introduced amino acid. Discussion In this paper we have described the protein folding, enzyme function, and thermostability of two novel rare MCAD mutants, R256T and K364R, which are strik- ingly different in their molecular behavior. The first mutation, R256T is a nonconservative substitution of a strongly basic internal residue by a smaller and less polar one. R256T was unaffected in its folding, with lev- els of tetramer formation in E. coli cells comparable to wild type, even at 37 °C. Likewise there was only a mod- erate decrease in the thermostability of this protein. Nevertheless, R256T is clearly an acute mutation, because, regardless of its stability, the catalytic activity was completely abolished, as determined by the ferrice- nium assay, ETF assay, and the INT ⁄ PES activity stain. As R256T was successfully purified by the same method as wild-type MCAD, i.e. using affinity elution with the substrate, this mutation is unlikely to impede acyl-CoA substrate binding, but rather must affect catalysis, either in the acceptance of reducing equivalents from the substrate, or the donation of these equivalents to ETF. Although K364R is a relatively conservative substi- tution, exchanging one basic residue for another, arginine is a bulkier residue, and may cause steric hin- drance of the local structure of helix J, or affect the interaction with neighboring helices of the C-terminal domain. K364R was found to be an acute, tempera- ture-sensitive folding mutation from the chaperonin studies. The mutant protein was, at most, moderately affected in its substrate kinetics, ETF interaction, and FAD affinity when compared with wild type. This suggests that, although K364R is acutely affected in its ability to fold into tetramer, whatever does fold correctly is functionally competent, though less stable than the wild type protein. The protein instability cau- ses the substrate to bind more loosely, as reflected in the K m and absorption characteristics. However this instability is not sufficient to impact greatly on the catalytic activity, as the k cat and the ETF interaction are relatively unaffected. This mutation seems to affect mainly the initial folding and stability of the tetramer, and similarly lowered levels of tetramer in human cells would account for the observed elevation of indicator acyl-carnitine levels. From the biochemical studies, it would appear that both R256T and K364R, although showing very differ- ent effects at the protein level, are both severe muta- tions in terms of their overall effect on expressed activity. As R256T has so far been found as a com- pound heterozygote with the K304E mutation, this could either mask or enhance the clinical manifestation of the disease [6,26]. Although the individuals with this mutation have remained asymptomatic, the analogous mutations in glutaryl-CoA dehydrogenase (R257W, compound heterozygote with P278S, and R257Q), are known to be disease-causing, indicating that R256T could also be potentially disease-causing [27]. As K364R was found as a homozygote, our biochemical studies are more directly applicable. Regardless of the enzyme activity as determined by biochemical testing, the actual outcome can vary from individual to individual depending on the functional overlap of VLCAD, LCAD, MCAD and SCAD, the efficiency of the chaperonin-aided folding, the effi- ciency of the detoxification of accumulated intermedi- ates, and avoidance of exposure to the environmental triggers. Certainly in the case of MCAD deficiency, environmental factors appear to outweigh the genetic factors [26]. The possibility that different mutations alone may cause varying severity of disease, resulting in the wide clinical manifestation, has been considered [28]. However, subsequent biochemical and molecular folding studies of the various point mutations have revealed no clear correlation between the genotype and phenotype [6]. This becomes most apparent in the study of the homozygous K304E, where the entire clin- ical spectrum of MCAD deficiency has been observed [5,6,26,29], suggesting that other background factors must modulate the severity of clinical presentation. Therefore there is no correlation evident between the effect of the mutations, as determined experimentally for the protein, and the severity of disease precipita- tion. Present evidence would suggest that, whilst the affected individuals in whom these new mutations were found have not shown overt clinical symptoms, these are nevertheless potentially dangerous mutations. MCAD catalysis (R256T) and folding (K364R) mutants L. P. O’Reilly et al. 4554 FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS Experimental procedures Chemicals 5-Bromo-4-chloro indol-3-yl phosphate ⁄ nitro blue tetrazo- lium (BCIP ⁄ NBT) tablets, octanoyl-CoA, 2,6-dichloro- phenolindophenol (DCPIP), INT, PES, ferrocene, sodium hexafluorophosphate were all obtained from Sigma-Ald- rich Ltd (Dorset, UK). Procion Red HE-3B textile dye was a generous gift from Dr C.V. Stead of the Dyestuffs Division of Imperial Chemical Industries (Blackley, Manchester, UK). Column chromatography media were supplied by Amersham Biosciences UK Ltd (Amersham, Buckinghamshire, UK). Filters for the Amicon Centricon device were purchased from Millipore Ireland BV (Cork, Ireland). Mutagenesis and subcloning of R256T and K364R The genes for the mutant MCAD proteins were overex- pressed in E. coli JM109 cells by using the pWT vector (encoding the mature part of the human MCAD gene, pre- ceded by an artificial methionine, under the control of the lac operon) in which the MCAD842GfiC (R256T) and MCAD1165AfiG (K364R) mutations were introduced by site-directed PCR-based mutagenesis [12,30]. Mutagenic antisense oligonucleotides for MCAD  842GfiC(5¢-GAT AAAACCACACCTGTAGTAGCTG-3¢) and MCAD 1165AfiG(5¢-CCTGTAGAAAGACTAATGAGGGATG CC-3¢) (mutagenic substitutions are shown in bold), and the antisense primer (5¢-GTAACGCCAGGGTTTTCCCA GTCAC-3¢) were used to generate a megaprimer. This was used in a secondary PCR, with the sense primer (5¢-GATC CAGATCCTAAAGCTCCTGCT-3¢), to generate the full- length fragment, which was then subcloned into the pWT vector, using the EcoRI and HindIII sites. The expression vectors were sequenced across the region encoding the MCAD gene, to exclude PCR-based errors. Each mutant MCAD vector was cotransformed into JM109 with either pGroESL (encoding the chaperones GroES and GroEL) [31] or pCap (empty vector control), and cultured as des- cribed elsewhere [12]. Polyacrylamide gel electrophoresis and western blotting SDS ⁄ PAGE, native PAGE, and western blotting were performed essentially as described previously, using BCIP ⁄ NBT tablets for colour development [9]. Protein purification The mutant protein and wild type were purified by anion exchange, and dye-affinity chromatography, as described elsewhere [32]. Activity staining This method was initially modified from an activity assay for short chain acyl-CoA dehydrogenase [33] by substitu- ting a tetrazolium dye acceptor for DCPIP. In the opti- mized protocol, native gels were submerged in the staining solution (50 mm glycine ⁄ NaOH, pH 9.6, 1 mm p-iodo- nitrotetrazolium violet, 10 mgÆmL )1 phenazine ethosulphate, 40 mm octanoyl-CoA) and placed on a shaker for approxi- mately 30 min until a strong colour developed. Stain development was arrested by rinsing the gel with H 2 O. Enzyme kinetics Reaction rates were measured with concentrations of octa- noyl-CoA from 1 lm to 100 lm. The reactions were carried out in 100 mm KP i ,5mm EDTA buffer, pH 7.6 at 25 °C with ferricenium as the final electron acceptor, as described elsewhere [34]. The results were analysed using Enzpack 3 software (Biosoft) to determine the K m and V max values by the Direct Linear [35] and Wilkinson [36] methods. The k cat was then determined, using the MCAD monomer M r values (46 590 for wild type, and 46 630 for K364R, the slight variation due to the mutation) to define the concentration of active sites. Michaelis–Menten plots were used only to display the results. Electron transferring flavoprotein (ETF) assay This assay utilizes 2,6-dichlorophenolindophenol as the final electron acceptor, in 50 mm KP i , 0.3 mm EDTA, 5% glycerol, pH 7.6 buffer, at 25 °C [37]. Thermal stability of enzyme activity in cleared bacterial lysates One hundred microlitre samples of bacterial lysates contain- ing mutant or wild-type MCAD (10 lgÆmL )1 in 100 mm KP i ,5mm EDTA, pH 7.6) were dispensed into separate Eppendorf flasks. Each was incubated for 10 min in a water bath at the chosen temperature before removing to ice, and sampling for activity [34] and western blot analysis [12]. Acknowledgements We warmly acknowledge the help and collaboration of Dr Simon Olpin of the Sheffield Children’s Hospital who detected the patient with the MCAD1166AfiG mutation and supplied material to make identification of this mutation possible. This work was supported by a grant from the March of Dimes Foundation (grant number 1-FY-2003–688 to BSA) and also by Grant 1C ⁄ 2002 ⁄ 073 under the International Collaboration Programme of Enterprise Ireland. L. P. O’Reilly et al. MCAD catalysis (R256T) and folding (K364R) mutants FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS 4555 References 1 Engel PC (1992) Acyl-CoA dehydrogenases. Chemistry and Biochemistry of Flavoenzymes (Muller F, ed), pp. 597–655. CRC Press, Boca Raton. 2 Thorpe C & Kim JJ (1995) Structure and mechanism of action of the acyl-CoA dehydrogenases. FASEB J 9, 718–725. 3 Schulz H (1991) Biochemistry of lipids, lipoproteins and membranes. Oxidation of Fatty Acids in Eukaryotes (Vance DE & Vance J, eds), pp. 87–110. Elsevier Science Publishers, Amsterdam. 4 Ding J & Roe CR (2001) Mitochondrial fatty acid oxi- dation disorders. The Metabolic and Molecular Basis of Inherited Disease (Scriver CR, Beaudet A, Sly WS & Valle D, eds), pp. 2297–2326. 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