Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism Matthias Mack 1 , Ute Schniegler-Mattox 2 , Verena Peters 3 , Georg F. Hoffmann 3 , Michael Liesert 4 , Wolfgang Buckel 4 and Johannes Zschocke 2 1 Institut fu ¨ r Technische Mikrobiologie der Hochschule Mannheim, Germany 2 Institut fu ¨ r Humangenetik, Ruprecht-Karls-Universita ¨ t Heidelberg, Germany 3 Abteilung fu ¨ r Allgemeine Pa ¨ diatrie, Ruprecht-Karls-Universita ¨ t Heidelberg, Germany 4 Labor fu ¨ r Mikrobiologie der Philipps-Universita ¨ t-Marburg, Germany In humans, isolated deficiencies of each of the six dif- ferent steps within the leucine degradation pathway (Fig. 1) cause their own characteristic disease [1]. The enzymes of this pathway are primarily located in the mitochondria. Together with the corresponding genes and their associated metabolic disorders they are sum- marized in Table 1. 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase (EC 4.2.1.18) catalyses the fifth step in the leucine degradation pathway, the reversible hydration of 3-MG-CoA to 3-hydroxy-3-methyl- glutaryl-CoA (HMG-CoA). Reduced or absent 3-MG- CoA hydratase activity causes a metabolic block (Fig. 1) and as a result, 3-MG-CoA accumulates within the mitochondrial matrix [2,3]. 3-MG-CoA is hydrolyzed in the mitochondrion by a yet unknown acyl-CoA hydrolase to form 3-methylglutaconic acid and free CoA, followed by export of 3-methylglutacon- ic acid from the mitochondrion. Reduced 3-MG-CoA hydratase activity also produces increased levels of 3-methylglutaric acid and 3-hydroxyisovaleric acid. Keywords leucine metabolism; 3-methylglutaconic aciduria type I; 3-methylglutaconyl- coenzyme A hydratase; AUH Correspondence M. Mack, Institut fu ¨ r Technische Mikrobiologie der Hochschule Mannheim, Windeckstr. 110, 68163 Mannheim, Germany Fax: +49 6212926420 Tel: +49 6212926496 E-mail: m.mack@hs-mannheim.de (Received 14 September 2005, revised 3 March 2006, accepted 7 March 2006) doi:10.1111/j.1742-4658.2006.05218.x The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is char- acterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that muta- tions in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V max ¼ 3.9 UÆmg )1 , K m ¼ 8.3 lm, k cat ¼ 5.1 s )1 ) and (E)-glutaconyl-CoA (V max ¼ 1.1 UÆmg )1 , K m ¼ 2.4 lm, k cat ¼ 1.4 s )1 ) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity. Abbreviations ARE, A + U-rich elements; Gct, glutaconate CoA-transferase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MBP, maltose binding protein; MGA (MGA1), 3-methylglutaconic aciduria (type I); 3-MG-CoA, 3-methylglutaconyl-CoA; MTP, mitochondrial trifunctional protein. 2012 FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 3-Methylglutaric acid is synthesized by the action of an as yet unspecified dehydrogenase on accumulating 3-MG-CoA (Fig. 1) whilst 3-hydroxyisovaleric acid is produced via the enzymatic hydration of 3-methylcro- tonyl-CoA (crotonase, EC 4.2.1.17) (Fig. 1) [2]. Conse- quently, humans with reduced or absent 3-MG-CoA hydratase activity show excessive urinary excretion of 3-methylglutaconic acid, 3-hydroxyisovaleric acid and Fig. 1. The metabolic pathway of (S)-leucine ( L-leucine) and isovalerate. Enzymes involved are as follows: 1, EC 2.6.1.42, branched chain amino transferase 1; 2, EC 1.2.4.4 ⁄ 2.3.1.168 ⁄ 1.8.1.4, branched chain 2-keto acid dehydrogenase complex; 3, EC 1.3.99.10, isovaleryl-CoA dehydrogenase; 4, EC 6.4.1.4, 3-methylcrotonyl-CoA carboxylase 1; 5, EC 4.2.1.18, 3-methylgluta- conyl-CoA hydratase; 6, EC 4.1.3.4, 3-hydroxy-3-methylglutaryl-CoA lyase; 7, EC 2.8.3.–, isovalerate-CoA-transferase; 8, crot- onase, EC 4.2.1.17. 9, unknown. Table 1. Enzymes, genes, and associated diseases of the human leucine degradation pathway. Enzyme name EC Gene OMIM Branched chain amino transferase 1 2.6.1.42 BCAT1 113520 Branched chain keto acid dehydrogenase E1, alpha ⁄ beta subunits 1.2.4.4 BCKDHA 608348 BCKDHB 248611 Dihydrolipoamide branched chain transacylase E2 2.3.1.168 DBT 248610 Dihydrolipoamide dehydrogenase E3 1.8.1.4 DLD 246900 Isovaleryl-CoA dehydrogenase 1.3.99.10 IVD 607036 3-Methylcrotonyl-CoA carboxylase 1 6.4.1.4 MCCC1 609010 3-Methylglutaconyl-CoA hydratase 4.2.1.18 AUH 250950 3-Hydroxy-3-methylglutaryl-CoA lyase 4.1.3.4 HMGCL 246450 M. Mack et al. Human 3-methylglutaconyl-CoA hydratase FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 2013 3-methylglutaric acid [2]. Human 3-MG-CoA hydra- tase deficiency is known as type I 3-methylglutaconic aciduria (MGA1, MIM 250 950). It has been found in association with variable phenotypes ranging from apparently normal development to severe psychomotor retardation with progressive neurological symptoms [4]. At present, three additional forms of MGA in humans have been recognized [5]. These diseases are not associated with reduced 3-methylglutaconyl-CoA hydratase levels and the excretion of 3-MG and 3-methylglutaric acid is secondary. Type II MGA (MIM 302060), also referred to as Barth syndrome, is a cardiomyopathy associated with neutropenia and growth retardation and caused by mutations in the gene encoding tafazzin (TAZ, previously denoted G4.5) [6]. Type III (MIM 258501) or Costeff syndrome, is a disorder caused by mutations in the OPA3 gene [7], leading to bilateral optic atrophy. Finally, type IV (MIM 250951) comprises a heteroge- neous group of patients with progressive neurological symptoms [5]. The molecular basis for type IV MGA is unknown, however, experiments with the fungus Aspergillus nidulans carrying null alleles in the known genes for 3-methylglutaconyl-CoA hydratase and 3-methylcrotonyl-CoA carboxylase strongly suggest that a second route for 3-MG biosynthesis exists [8]. Interestingly, certain patients with Smith–Lemli–Opitz syndrome also show abnormally increased plasma levels of this compound, further challenging our under- standing of 3-methylglutaconic acid metabolism [9]. Lastly, pregnancy was reported as a possible cause of MGA [10]. For a long time it had been unclear which enzyme was responsible for the hydratase step within leucine degradation. A 3-MG-CoA hydratase was partially purified from bovine ⁄ ovine liver [11]. It was established that this enzyme catalyses the syn-addition of water to (E)-3-MG-CoA leading to (S)-HMG-CoA [12]. Another enoyl-CoA hydratase, mitochondrial crotonase, is not active using HMG-CoA and measur- ing the reverse (dehydration) reaction [13]. Mitochond- rial trifunctional protein (MTP) is the main enoyl-CoA hydratase in long chain fatty acid b-oxidation [14]. This enzyme, however, is unlikely to be involved in leucine degradation since MTP deficiency (MIM 143450, MIM 600890) is not associated with increased urinary excretion of 3-methylglutaconic acid. A protein was purified from human brain cells by affinity chro- matography using the immobilized RNA-oligonucleo- tide (AUUUA) 5 or ‘AU’ followed by cloning of the corresponding gene [15]. Interestingly, the gene showed sequence similarity to enoyl-CoA-hydratases-1 (2- trans-enoyl-CoA-hydratases; EC 4.2.1.17) and its gene product had weak enoyl-CoA-hydratase activity using crotonyl-CoA as a substrate [15]. The gene encoding this bifunctional protein was named AUH (‘AU bind- ing homolog of enoyl-CoA hydratase’). The RNA- binding activity of the human protein and also of the murine homologue was investigated further, its biologi- cal function, however, remained unclear [16,17]. The three-dimensional structure of AUH was determined at 2.2 A ˚ resolution and regarding its hydratase activity a high affinity for short-chain substrates was predicted [18]. The first pure preparation of a 3-MG-CoA hydra- tase was obtained from the bacterium Acinetobacter sp. which aerobically grows on isovalerate as sole car- bon and energy source. Isovalerate is activated by a CoA-transferase (2.8.3.–) to give isovaleryl-CoA (Fig. 1). Isovalerate is metabolized via isovaleryl-CoA, an intermediate of the oxidative (S)-leucine degrada- tion pathway [19]. The gene for 3-MG-CoA hydratase in Acinetobacter sp. was partially cloned. The transla- ted nucleotide sequence had weak similarities to enoyl- CoA-hydratases (30% identity) and also human AUH. It was shown by two independent groups, that MGA1 patients with reduced or absent hydratase activity have mutations within the AUH gene [13,20]. In addition it was shown that AUH has 3-MG-CoA hydratase activity using HMG-CoA as a substrate and measuring the dehydration reaction. AUH locates on chromosome 9q22.31. The present work was initiated to kinetically charac- terize AUH on its presumed natural substrate 3-MG- CoA using a new strategy for its synthesis and developing a new assay. In addition, a mutant form of AUH (A240V) derived from an MGA1 patient was tested using 3-MG-CoA. Results Overexpression of AUH in Escherichia coli and purification of the corresponding gene product The gene for AUH which was cloned from a cDNA library by Nakagawa et al. [15] encodes 339 amino acids specifying a 40-kDa protein (AUHp40). Western blot analysis of brain extracts consistently revealed a 32 kDa AUH protein and it was thus assumed that the mature form of human AUH in brain has a molecular weight of 32 kDa (AUHp32) [15]. For the kinetic characterization of AUH described in the work at hand, AUH was overproduced in Escherichia coli as a maltose binding protein fusion (MBP-AUH). The complete AUH gene (producing MBP-AUHp40 in E. coli) but also a truncated form of AUH (producing MBP-AUHp32 in E. coli) were ligated into the Human 3-methylglutaconyl-CoA hydratase M. Mack et al. 2014 FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS bacterial expression vector pMAL-c2. Consequently, two different forms of AUH, namely MBP-AUHp40 and MBP-AUHp32 could be isolated from the corres- ponding E. coli strains. MBP-AUHp40 and MBP- AUHp32 were purified to apparent homogeneity. In a subsequent step, the MBP portion of both fusion pro- teins was removed by proteolysis and the resulting pro- teins AUHp40 and AUHp30 were again purified by chromatography. Thus, four different pure fractions of AUH could be generated: MBP-AUHp40, AUHp40, MBP-AUHp32 and AUHp32. Using the substrate 3-MG-CoA no difference in enzymatic activity was detected between the four AUH forms MBP-AUHp40, AUHp40, MBP-AUHp32 and AUHp32. Since the purification procedure for MBP-AUHp40 was highly reproducible, the kinetic data were collected using purified MBP-AUHp40. Enzymatic synthesis of 3-MG-CoA and glutaconyl-CoA The substrates 3-MG-CoA and glutaconyl-CoA were synthesized using recombinant glutaconate CoA-trans- ferase from the glutamate fermenting bacterium Acid- aminococcus fermentans [21,22]. This enzyme catalyses the transfer of coenzyme A from a CoA-donor to a CoA-acceptor (Fig. 2). In addition to its natural sub- strate (R)-2-hydroxyglutarate, glutaconate CoA-trans- ferase uses glutaconate, 3-methylglutaconate and other short chain carboxylic acids as CoA-acceptors. Because the CoA-acceptor 3-methylglutaconate was not com- mercially available, it was produced by alkaline hydroly- sis of the corresponding dimethylester. HPLC analysis of the enzymatically produced 3-MG-CoA revealed five signals (Fig. 3). The compounds producing the signals were analyzed by mass spectrometry. The compounds producing the first two signals (peak 1 and peak 2) had molecular masses corresponding to unreacted acetyl- CoA and free coenzyme A. The compounds producing the following signals (peak 3, peak 4 and peak 5) were found to all have the same relative molecular mass of 893 matching the calculated molecular mass of 3-MG- CoA (893.647). Thus, three 3-MG-CoA isomers were produced using the enzyme glutaconate CoA-transferase (Fig. 4). The three different forms of 3-MG-CoA were separated by HPLC, collected and their concentration was determined using an enzymatic 5,5¢-dithiobis-2-ni- trobenzoate-based assay. Subsequently, the 3-MG-CoA isomers were tested using AUH (Fig. 3). It was found, that peak 5 (2 mm) was readily converted to (S)-HMG- CoA. In addition, free CoA was detected. Peak 4 (2 mm) produced significantly less HMG-CoA and also, in this reaction, a substantial amount of free CoA was found. Peak 3 (0.5 mm) gave mainly free CoA and only small amounts of HMG-CoA. Peak 5, being the best sub- strate, should correspond to (E)-3-MG-1-CoA, the inter- mediate of the leucine degradation pathway (Fig. 4). Peak 4 is most likely to correspond to (E)-3-MG-5-CoA. Peak 3 is probably (Z)-3-MG-5-CoA. Glutaconyl-CoA was prepared accordingly. Also in this reaction two compounds were produced by glut- aconate CoA-transferase. The molecules were separ- ated by HPLC, analyzed by mass spectrometry and were found to both have the same relative molecular mass of 881 corresponding to glutaconyl-CoA (881.247). Peak 1 was dominant and most likely was glutaconyl-1-CoA. Peak 2 probably was glutaconyl-5- CoA. The two isomers were separated from each other. However, upon repeated analysis of the isolated compounds, the same two signals appeared. The two isomers seem to interconvert into each other making a separation impossible. Therefore, a mixture of the two isomers had to be used in the following studies. Kinetic constants for AUH on different CoA-substrates Besides (E)-3-MG-1-CoA, the potential substrates glu- taconyl-CoA and HMG-CoA as well as crotonyl-CoA, 3-hydroxybutyryl-CoA and 3-methylcrotonyl-CoA were used for the kinetic characterization of AUH. The data are summarized in Table 2. Overexpression of a mutant form of AUH and its activity on (E)-3-MG-1-CoA Mutations in AUH are linked to the metabolic disease MGA1. Most published patients have been homozy- gous or compound heterozygous for null mutations expected to completely remove protein function [13,20]. One patient was compound heterozygous for a null mutation and a missense mutation A240V (c.719C>T). This mutant form of AUH was overproduced as an Fig. 2. General mechanism for coenzyme A-transferases. The CoAS – moiety is transferred from the carboxyl group of the CoA-donor (R 1 -COO – ) to the carboxyl group of the CoA-acceptor (R 2 -COO – ). In the case of glutaconate CoA-transferase from A. fermentans, CoAS – transiently is bound to the c-carboxyl group of bE54 [24]. M. Mack et al. Human 3-methylglutaconyl-CoA hydratase FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 2015 MBP fusion (MBP-AUHp40 mut )inE. coli according to the wild-type enzyme and tested using the substrate (E)-3-MG-1-CoA. In these experiments, the specific activity of MBP-AUHp40 mut was 9% (0.068 UÆmg )1 protein) in comparison to the wild-type enzyme (0.76 UÆmg )1 protein). Hence, the mutation A240V cau- ses a significant loss of enzyme activity. Direct nonisotopic assay of 3-MG-CoA hydratase in cultured human skin fibroblasts The nonisotopic 3-MG-CoA hydratase assay that was developed during this work was evaluated for its use in testing homogenates derived from human skin fibro- blast cultures. Different cell cultures derived from type I MGA patients and from wild-type controls were grown, fibroblast homogenates were prepared and tes- ted using 3-MG-CoA (10 lm). It was not possible to detect HMG-CoA in this assay probably due to rapid further processing of this common intermediate by enzymes present in the fibroblast homogenate (e.g. by 3-hydroxy-3-methylglutaryl-CoA lyase; EC 4.1.3.4). Therefore, 3-MG-CoA (10 lm) was replaced by gluta- conyl-CoA (10 lm), which had been shown in our work to be an excellent substrate for AUH. The product of this reaction, 3-hydroxyglutaryl-CoA, was mAU 0 200 400 600 0 14 16 18 20 22 24 min Acetyl CoA CoA (Z)-3-methylglutaconyl-5-CoA MW 893,36 (E)-3-methylglutaconyl-5-CoA MW 893,36 (E)-3-methylglutaconyl-1-CoA MW 893,59 0 200 400 600 1000 mAU HMG-CoA 800 CoA (E)-3-methyl- glutaconyl-1-CoA AUHAUHAUH 0 10 20 min 0 20 40 60 80 100 120 mAU CoA (Z)-3-methyl- glutaconyl- 5-CoA 010 20 min 200 400 600 800 1000 mAU 0 010 20 min CoA HMG-CoA (E)-3- methyl- glutaconyl- 5-CoA 1 2 3 4 5 HMG-CoA B A Fig. 3. Isomers of 3-MG-CoA as substrates for human 3-MG-CoA hydratase (AUH). (A) 3-MG-CoA was enzymatically synthesized by incuba- ting 100 m M 3-methylglutaconate in 100 mM potassium phosphate pH 7.0 with 1 mM acetyl-CoA (reaction volume 1 mL). Synthesis was started by addition of 0.25 mg glutaconate-CoA-transferase from A. fermentans. The reaction was analyzed by HPLC and the CoA deriva- tives were detected by their absorbance at 260 nm. Five signals were found, analyzed by mass spectrometry and assigned to be free CoA (peak 1), acetyl-CoA (peak 2) (Z)-3-MG-5-CoA (peak 3) (E)-3-MG-5-CoA (peak 4) and (E)-3-MG-1-CoA (peak 5). The determined relative molec- ular masses (MW) of the 3-MG-CoA compounds producing the signals are shown. Acetyl-CoA was purchased from Sigma Aldrich (A 2056) and does contain traces of free CoA (peak 1). Nothing is known about the fronting and the peak shoulder of peak 1, however, the compound is described by the supplier as only approximately 95% pure. The tailing of acetyl-CoA (peak 2) most likely also is due to impurities of the commercially available compound. If acetyl-CoA (Sigma Aldrich A 2056) only (without the addition of fibroblast homogenate) is applied to the HPLC-system the same picture appears. Thus, it seems, that the fronting, the shoulder and the tailing is due to acetyl-CoA and not due to any other compound. (B) The peaks 3, 4, and 5 were isolated by HPLC and used as substrates for AUH. The AUH assay contained 2 m M of the respective isomers of 3-MG-CoA in a total volume of 25 lL. The reaction was started by addition of AUH (1 lg), incubated for 1 h and the CoA products were HPLC-detected by their absorbance at 260 nm. Peak 3 produced small amounts of HMG-CoA and large amounts of free CoA. Peak 4 produced HMG-CoA and also large amounts of free CoA. Peak 5 produced large amounts of HMG-CoA, but also free CoA. Human 3-methylglutaconyl-CoA hydratase M. Mack et al. 2016 FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS readily detectable (Fig. 5). We investigated fibroblast homogenates from two controls and fibroblast homo- genates from three patients with established MGA type 1. Patient 1 [20] was homozygous for a mutation leading to a stop codon at residue 197 (R197X) and patient 2 [20] was homozygous for a mutation at the splice acceptor site of intron 8 (IVS8–1G>A). Patient 3 [20] was compound heterozygous for a missense mutation A240V (c.719C>T) in exon 7 and an inser- tion mutation c.613–614insA. This insertion causes a frameshift that starts at Met205 and leads to the intro- duction of a stop codon after four amino acids. The intra-assay variation, estimated by measuring four fibroblast homogenates in a single experiment, was 3.9%, the interassay variation was 5.4% (n ¼ 3 days). The fibroblast material from all MGA1 patients pro- duced significantly less (4–16 mUÆmg )1 protein, mean ¼ 8mUÆmg )1 protein) of 3-hydroxyglutaryl-CoA as compared to the two controls (72 mUÆmg )1 protein and 80 mUÆmg )1 protein). These results show, that the test measuring the hydratase reaction of AUH indeed is useful for the direct analysis of fibroblast cultures derived from patients. The residual activity within the patient material may be due to other enzymes in the fibroblast protein mixture. No other specific soluble human enzyme, however, is known to accept glutaco- nyl-CoA as a substrate and to produce 3-hydroxyglut- aryl-CoA. Fig. 4. Possible isomeric products of 3-MG-CoA produced by recombinant glutaconate CoA-transferase (Gct) from A. fermentans. Gct was used to produce 3-MG-CoA from (E,Z)-3-methylglutaconate and acetyl-CoA. (A) Gct transfers CoAS – to either the 1-carboxyl- (left) or the 5-carboxyl group (right) of (E)-3-methylglutaconate. (E)-3-MG-1-CoA (left) was the best substrate for human 3-MG-CoA hydratase (AUH). According to this scheme (E)-3-MG-5-CoA (right) bound to Gct isomerizes to give (E)-3-MG-1-CoA. (B) The production of (Z)-3-MG-5-CoA by Gct is probably due to the possible trans-conformation of the 5-carboxyl group of (Z)-3-methylglutaconate. Table 2. Kinetic constants of AUH (human 3-methylglutaconyl-CoA hydratase). Substrate K m (lM) V max a (UÆmg )1 ) k cat (s )1 ) k cat ⁄ K m (lM )1 Æs )1 ) (E)-3-Methylglutaconyl-1-CoA 8.3 3.9 5.1 0.6 (R,S)-3-Hydroxy-3- methylglutaryl-CoA 2250 0.2 0.26 1.2 )4 (E)-Glutaconyl-CoA 2.4 1.1 1.4 0.6 Crotonyl-CoA 12100 5.2 6.8 5.6 )4 3-Hydroxybutyryl-CoA 55200 1.3 1.7 3.1 )5 3-Methylcrotonyl-CoA 347 2.2 2.9 8.2 )3 a Specific activities (UÆmg )1 ) are in lmolÆmin )1 x mg protein. M. Mack et al. Human 3-methylglutaconyl-CoA hydratase FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 2017 Discussion In the present study, we characterized the 3-MG-CoA hydratase reaction of leucine catabolism at the protein and DNA levels and developed a novel assay for enzyme analysis in a diagnostic setting. The human AUH protein was first recognized by its ability to bind A + U-rich elements (ARE) of mRNA. Surprisingly, AUH showed sequence similarity to enoyl-CoA hydra- tases, suggesting that this enzyme had another function in cellular metabolism. Indeed, AUH had enoyl-CoA hydratase activity, which was described as an additional intrinsic function of the protein. Its role in intermediary metabolism, however, was not clear [15]. It was subse- quently shown, that the metabolic disorder MGA1 caused by reduced 3-MG-CoA hydratase activity is associated with mutations in the AUH gene. This indi- cated that AUH, in addition to its RNA binding func- tion, must play an important role in leucine catabolism. AUH was overproduced in E. coli and characterized by measuring the reverse reaction, the dehydration of HMG-CoA to 3-MG-CoA. The reaction was followed photometrically [4]. In order to measure AUH in the forward reaction (hydratase activity), it was necessary to synthesize 3-MG-CoA, which is not commercially available. Glut- aconate CoA-transferase (Gct) from A. fermentans proved to be useful for the enzymatic production of this compound. Earlier, Gct was reported to be specific for (E)-glutaconate and to be completely inactive with (Z)-glutaconate [21]. The activity of Gct using the CoA-donor acetyl-CoA and the CoA-acceptor 3-meth- ylglutaconate was relatively low. Our data suggest that Gct produces three isomers. The production of (Z)-3-MG-5-CoA is probably due to the possible trans-conformation of the C 5 -carboxyl group of (Z)-3- methylglutaconate (Fig. 4). Most of (Z)-3-MG-5-CoA was hydrolyzed by AUH to give free CoA and (Z)-3- methylglutaconate, trace amounts of HMG-CoA, however, were detected. (E)-3-MG-5-CoA was a better substrate for hydration with AUH. An explanation for this may be, that upon binding to AUH, (E)-3-MG-5- CoA is isomerized to give (E)-3-MG-1-CoA (Fig. 4A). An intrinsic isomerase activity has also been reported for 4-hydroxybutyryl-CoA-dehydratase of Clostridium aminobutyricum [23]. The isomerization reaction, how- ever, obviously takes time and the acyl-CoA-hydrolase reaction is favored over the hydratase reaction produ- cing free CoA and (E)-3-methylglutaconate. The best substrate for AUH was (E)-3-MG-1-CoA (K m ¼ 8.3 lm, V max ¼ 3.9 UÆmg )1 , k cat ¼ 5.1), which is the intermediate of the leucine degradation pathway. Surprisingly, also with this substrate, large amounts of free CoA were produced. Enzyme assays for the mAU 0 20 40 60 80 100 120 140 010 20 min 1 4 AB C mAU 0 20 40 60 80 100 120 140 mAU 0 20 40 60 80 100 120 140 0 10 20 min 1 2 3 4 0 10 20 min 1 2 3 4 glutaconyl-CoA glutaconyl- CoA 3-hydroxy- glutaryl-CoA 3-hydroxy- glutaryl- CoA glutaconyl- CoA Fig. 5. Direct nonisotopic assay of 3-MG-CoA hydratase (AUH) in cultured human skin fibroblasts. 3-MG-CoA hydratase was tested in fibro- blast homogenates using glutaconyl-CoA (10 l M) as a substrate. The reaction was started by the addition of fibroblast homogenate (55 mg fibroblast proteinÆL )1 ), incubated for 1 h and the products of the reaction were HPLC-detected by their absorbance at 260 nm. (A) As a con- trol, the assay mixture was incubated without the addition of fibroblast homogenates. Two different cell cultures derived from a wild-type control (B) and an MGA1 patient (C, homozygous for mutation IVS8-1G>A in the AUH gene) were grown and fibroblast homogenates were prepared in phosphate-buffered saline (55 mg proteinÆmL )1 ). The compounds producing the signals (peak 1, peak 2, peak 3 and peak 4) were analyzed by mass spectrometry. Peak 1 is free CoA, peak 2 probably is glutaryl-CoA, peak 3 is 3-hydroxyglutaryl-CoA and peak 4 is the sub- strate glutaconyl-CoA. The fibroblast homogenate derived from the MGA1 patient produces significantly less (9%) of 3-hydroxyglutaryl-CoA confirming AUH deficiency. Human 3-methylglutaconyl-CoA hydratase M. Mack et al. 2018 FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS characterization of AUH have been carried out with crotonyl-CoA as a substrate or by measuring the dehy- dratase reaction using HMG-CoA. In these experi- ments, an acyl-CoA-hydrolase activity of AUH was not detected. This is the first report showing kinetic data for purified AUH, although a 3-MG-CoA hydra- tase activity was found earlier in fibroblast and lym- phocyte lysates measuring the hydration reaction [3]. At that time, the substrate [5- 14 C]3-MG-CoA was pre- pared by incubation of 3-methylcrotonyl-CoA with 3-methylcrotonyl-CoA-carboxylase in the presence of NaH 14 CO 3 . In this work, K m values for the hydration of [5- 14 C]3-MG-CoA of 6.9 lm (fibroblast) and 9.4 lm (lymphocyte), respectively, were reported [3]. The formation of [5- 14 C]3-methylglutaconate from [5- 14 C]3- methylglutaconyl-CoA was interpreted as nonspecific hydrolysis. Our results suggest that CoA-hydrolysis is an intrinsic function of AUH. Experiments with the substrate HMG-CoA and AUH showed that the hydration reaction (k cat ¼ 5.1) is favored by a factor of 20 over the dehydration reac- tion (k cat ¼ 0.26) which is consistent with the main role of AUH in leucine catabolism, the hydration of 3-MG-CoA. Comparing the turnover numbers of glu- taconyl-CoA (k cat ¼ 1.4) and 3-MG-CoA (k cat ¼ 5.1) it is obvious, that 3-MG-CoA is a better substrate. Crotonyl-CoA (k cat ¼ 6.8) and 3-hydroxybutyryl-CoA (k cat ¼ 1.7) have higher K m values (12 and 55 mm), indicating that the missing carboxylate reduces affinity to the active site. Also in this case, the hydration reac- tion was favored over the dehydration reaction (factor of 4). The mutant enzyme MBP-AUHp40 mut (A240V), identified in one MGA1 patient, had a clearly reduced 3-MG-CoA hydratase activity (9% of the wild-type enzyme). This finding provides further evidence con- firming that AUH is indeed the main hydratase in the human leucine degradation pathway and that muta- tions leading to reduced hydratase activity are respon- sible for the MGA1 phenotype. The need to differentiate patients with AUH defi- ciency from patients with other forms of MGA requires the availability of a sensitive and specific enzyme assay. Our data show that the hydratase reaction of AUH is favored over the dehydratase reaction (factor of 20). Hence, measuring the for- ward reaction in fibroblast homogenates of patient- derived cells should increase the sensitivity of an AUH test. The product of this reaction, however, is the common intermediate HMG-CoA, which is quickly degraded by, e.g. 3-hydroxy-3-methylglutaryl- CoA lyase (EC 4.1.3.4), to give acetyl-CoA and acetoacetate. As glutaconyl-CoA is a very good substrate for AUH and since the product of the hy- dratase reaction, 3-hydroxyglutaryl-CoA, is not an intermediate within human metabolism, we hypothes- ized that glutaconyl-CoA may be used as a substrate for testing AUH activity in a routine setting. Indeed, we were able to show that AUH activity in fibro- blasts can be determined by monitoring the forma- tion of 3-hydroxyglutaryl-CoA. The production of a small amount of 3-hydroxyglutaryl-CoA in a patient homozygous for a null mutation in the AUH gene may be due to the action of another mitochondrial hydratase, e.g. crotonase. This will need to be taken into consideration when the assay is used in a diag- nostic setting. Nevertheless, we believe that the novel assay may be a superior method for confirmation of AUH deficiency in fibroblast homogenates. In summary, our data show that the main biological function of AUH in human metabolism is the hydra- tion of (E)-3-MG-CoA to (S)-HMG-CoA in the leu- cine degradation pathway. Experimental procedures Production and purification of human AUH in E. coli The production of AUHp40 (precursor form), AUHp32 (mature form), AUHp40A240 V and AUHp32A240 V in E. coli was performed using the pMAL-c2 bacterial expres- sion vector [17]. The different forms of AUH were pro- duced as fusions to the MBP of E. coli. The purification of the gene products was carried out as previously described [17]. Protein was estimated using the method of Bradford [24]. Production and purification of glutaconate CoA- transferase from A. fermentans in E. coli The production of glutaconate CoA-transferase from A. fermentans in E. coli and its subsequent purification was carried out as described earlier [22]. Site-directed mutagenesis Plasmids corresponding to constructs a and b [17] were modified using the Stratagene QuikChange Site-Directed Mutagenesis Kit and the mismatch oligonucleotides AUH FW 5¢-AGCTCATATTCTCTGTGCGAGTCCTCGATG GC-3¢ and AUH RP 5¢-GCCATCGAGGACTCGC ACA GAGAATATGAGCT-3¢ (the c.719C>T mutation leading to the amino acid exchange A240V is underlined). The AUH genes were proof-sequenced and no secondary muta- tions were detected. M. Mack et al. Human 3-methylglutaconyl-CoA hydratase FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 2019 Mass spectrometry The CoA-esters were separated by HPLC, ionized by ESI and detected by TOF. The HPLC system consisted of a HP1100 series binary-gradient pump, a vacuum degasser (all from Hewlett-Packard), and a CTC HTS PAL autosampler (CTC). The dry sample was dissolved in water and 20 lL injected onto a 4 · 40-mm Grom-Sil120 ODS-4 HE column (3-lm particle diameter; Grom). The samples were separated from interfering compounds by a gradient between solution B (acetonitrile +1 vol% formic acid) and solution A (water + 1 volume % formic acid). The gradient (1 mLÆmin )1 ) was as follows: 0–5 min, 0% B to 83% B; 6–8 min, 100% B. All gradient steps were linear, and the total analysis time, inclu- ding equilibration, was 10 min. A splitter between the HPLC column and the mass spectrometer was used, and 100 lLÆmin )1 of eluent was introduced into the mass spec- trometer. A LCT TOF (time-of-flight) mass spectrometer (Micromass) was used in the negative and positive electro- spray ionization (ESI) mode. Nitrogen was used as the neb- ulizing gas. The capillary voltage was 3 kV, the source temperature was set at 120 °C, and the optimal cone-voltage energy was 45 V. Enzymatic synthesis of 3-MG-CoA and glutaconyl-CoA Alkaline hydrolysis of dimethyl (E,Z)-3-methylglutaconate (Sigma-Aldrich, Deisenhofen, Germany) in 1 m NaOH for 30 min under reflux followed by exchange of Na + against H + with the ion exchanger Dowex 50 W · 8(H + -form, Serva, Heidelberg, Germany) yielded (E,Z)-3-methylgluta- conic acid. Its CoA-derivative 3-MG-CoA was enzymatically synthesized by incubating 100 mm 3-methylglutaconate in 100 mm potassium phosphate pH 7.0 with 1 mm acetyl- CoA. The reaction (1 mL) was started by addition of 0.25 mg recombinant glutaconate CoA-transferase (EC 2.8.3.12) from A. fermentans which was purified from an overproducing E. coli strain [22]. After 1 h at room tempera- ture the reaction was stopped by addition of an equal vol- ume of 8 m guanidinium chloride (1 mL) and the pH was adjusted to 3–4 using 1 m HCl. The desired product 3-MG- CoA was separated from unreacted compounds using a Waters Sep – Pak tC18 column (1 mL cartridge, 100 mg sorbent) (Waters, Eschborn, Germany). The column was first treated with 10 volumes 0.1% (v ⁄ v) trifluoroacetic acid in H 2 O, 50% acetonitrile (v ⁄ v). The acetonitrile component also contained 0.1% trifluoroacetic acid. Subsequently, the column was washed with 10 volumes 0.1% (v ⁄ v) trifluoro- acetic acid in H 2 O. The samples were loaded and the column was washed with 10 volumes 0.1% (v ⁄ v) trifluoroacetic acid in H 2 O. The CoA-ester was eluted with 10 volumes 0.1% (v ⁄ v) trifluoroacetic acid in H 2 O, 50% acetonitrile (v ⁄ v) con- taining 0.1% trifluoroacetic acid. After evaporation in vacuo, the CoA-ester was dissolved in water and stored at )20 °C. For analytic and preparative purposes a Phenomenex Syn- ergi 4 l Polar-RP 80 A column (5 lm) was used at a flow rate of 1 mLÆmin )1 (Phenomenex, Aschaffenburg, Ger- many). The eluents were 0.1% trifluoroacetic acid (v ⁄ v) in H 2 O (solution A) and 0.085% trifluoroacetic acid (v ⁄ v) in acetonitrile (solution B). The columns were equilibrated for 5 min with solution A. After injection of the CoA derivative a linear gradient was applied from 0 to 8% solution B within 3 min in order to remove impurities. The separation was achieved with a gradient from 8 to 13% solution B within 20 min. Afterwards the column was regenerated with 100% solution B for 15 min followed by 100% sol A for 10 min. The CoA esters were detected by their absorbance at 260 nm, evaporated in vacuo and dissolved in H 2 O. The CoA esters were identified by their masses using mass spectro- metry. The concentration of 3-MG-CoA and glutaconyl- CoA was determined enzymatically in a cuvette (1 mL) containing 100 mm potassium phosphate pH 7.0, 200 mm sodium acetate, 1 mm 5,5¢-dithiobis-2-nitrobenzoate and 1mm oxaloacetate, k ¼ 412 nm, e ¼ 14.2 mm )1 Æcm )1 [22,25]. An increase in absorbance after addition of 3-MG-CoA was due to free CoASH. If acetyl-CoA was present, a further increase followed the addition of 10 lg citrate synthase (Roche, Mannheim, Germany). The final increase after addi- tion of 10 lg glutaconate CoA-transferase was proportional to the concentration of 3-MG-CoA. The CoA-substrate glutaconyl-CoA was prepared accordingly from glutaconic acid (Fluka 49360) and acetyl-CoA. Other CoA-substrates (R,S)-HMG-CoA, crotonyl-CoA, 3-methylcrotonyl-CoA and 3-hydroxybutyryl-CoA were obtained from Sigma- Aldrich. Assay of 3-MG-CoA hydratase The assay contained in a total volume of 25 lL50mm Tris HCl pH 7.4, 10 mm EDTA, 1 mgÆmL )1 bovine serum albu- min and 0.05–0.2 mm 3-MG-CoA. The reaction was started by addition of the enzyme (1 lg). The products of the reac- tion were analyzed by HPLC as described above and mass spectrometry. The kinetic constants K m (lM) and V max (UÆmg protein )1 ) were evaluated with the Michaelis-Menten equation and Lineweaver-Burk plots using the Microsoft Excel program. The turnover numbers, k cat (s )1 ), were calculated with the subunit molecular mass (78.4 Da) of MBP-AUHp40. Direct nonisotopic assay of 3-MG-CoA hydratase in cultured human skin fibroblasts Fibroblasts were grown and harvested as described elsewhere [26]. Cells were suspended in 200 lL phosphate-buffered Human 3-methylglutaconyl-CoA hydratase M. Mack et al. 2020 FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS saline by repeated pipetting and sonicated three times on ice for 15 s at 8 W at 45-s intervals. An aliquot of the fibroblast homogenate (55 mgÆL )1 fibroblast protein) was added to the 3-MG-CoA hydratase assay. Protein was estimated using the method of Bradford [24]. The 3-MG-CoA hydratase assay mixture contained, in a final volume of 25 lL, 100 mm Tris- HCl (pH 8.0), 10 mm EDTA, 1 gÆL )1 bovine serum albumin and 10 lm 3-MG-CoA or glutaconyl-CoA. After incubation at 37 °C for 60 min, the reaction was terminated by the addi- tion of 2.5 lLof2m HCl. The samples were homogenized, and the assay tubes were placed on ice. After 5 min, the homogenates were brought to pH 6 with 2 mm KOH, 1 mm Mes (pH 6) and centrifuged at 21 000 g for 10 min at 4 °C. The supernatant was transferred to an HPLC vial. The products of the reaction were detected at 260 nm using the HPLC system described above for the synthesis of the CoA- esters. The assay was linear with an incubation time up to at least 60 min with up to 70 mgÆL )1 total protein. Acknowledgements We are grateful to C. Moroni and J. Nakagawa for sharing the cDNA encoding human AUH. This work received financial support from the Deutsche Fors- chungsgemeinschaft, Grant number Zs 17 ⁄ 4–2. References 1 Sweetman L & Williams JC (2001) Branched chain organic acidurias. The Metabolic and Molecular Bases of Inherited Disease (Scriver, C R, Beaudet, A L, Sly, W S & Valle, D, eds), pp. 2125–2163. McGraw-Hill, New York. 2 Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D & Wadman SK (1982) Inherited 3-methylglutaconic aciduria in two brothers – another defect of leucine metabolism. J Pediatr 101, 551–554. 3 Narisawa K, Gibson KM, Sweetman L, Nyhan WL, Duran M & Wadman SK (1986) Deficiency of 3-methyl- glutaconyl-coenzyme A hydratase in two siblings with 3-methylglutaconic aciduria. J Clin Invest 77, 1148–1152. 4 Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, Enoki M, Watanabe H & Takada G (1999) 3-Methyl- glutaconic aciduria type I: clinical heterogeneity as a neurometabolic disease. J Inherit Metab Dis 22, 1–8. 5 Gibson KM, Elpeleg ON, Jakobs C, Costeff H & Kelley RI (1993) Multiple syndromes of 3-methylglutaconic aciduria. Pediatr Neurol 9, 120–123. 6 Bione S, D’Adamo P, Maestrini E, Gedeon AK, Bolhu- is PA & Toniolo D (1996) A novel X-linked gene, G4.5. is responsible for Barth syndrome. Nat Genet 12, 385– 389. 7 Anikster Y, Kleta R, Shaag A, Gahl WA & Elpeleg O (2001) Type III 3-methylglutaconic aciduria (optic atrophy plus syndrome, or Costeff optic atrophy syndrome): identification of the OPA3 gene and its founder mutation in Iraqi Jews. Am J Hum Genet 69, 1218–1224. 8 Rodriguez JM, Ruiz-Sala P, Ugarte M & Penalva MA (2004) Fungal metabolic model for type I 3-methylgluta- conic aciduria. J Biol Chem 279, 32385–32392. 9 Kelley RI & Kratz L (1995) 3-Methylglutaconic acide- mia in Smith–Lemli–Opitz syndrome. Pediatr Res 37, 671–674. 10 Walsh R, Conway H, Roche G, Naughten E & Mayne PD (1997) 3-Methylglutaconic aciduria in pregnancy. Lancet 349, 776. 11 Hilz H, Knappe J, Ringelmann E & Lynen F (1958) Methylglutaconase, eine neue Hydratase, die am Stoff- wechsel verzweigter Carbonsa ¨ uren beteiligt ist. Biochem Z 329, 476–489. 12 Messner B, Eggerer H, Cornforth JW & Mallaby R (1975) Substrate stereochemistry of the hydroxymethyl- glutaryl-CoA lyase and the methylglutaconyl-CoA hydratase reactions. Eur J Biochem 53, 255–264. 13 Ijlst L, Loupatty FJ, Ruiter JP, Duran M, Lehnert W & Wanders RJ (2002) 3-Methylglutaconic aciduria type I is caused by mutations in AUH. Am J Hum Genet 71, 1463–1466. 14 Wanders RJLIJ, Poggi F, Bonnefont JP, Munnich A, Brivet M, Rabier D & Saudubray JM (1992) Human trifunctional protein deficiency: a new disorder of mito- chondrial fatty acid beta-oxidation. Biochem Biophys Res Commun 188, 1139–1145. 15 Nakagawa J, Waldner H, Meyer-Monard S, Hofsteenge J, Jeno P & Moroni C (1995) AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl- CoA hydratase activity. Proc Natl Acad Sci USA 92, 2051–2055. 16 Brennan LE, Nakagawa J, Egger D, Bienz K & Moroni C (1999) Characterisation and mitochondrial localisa- tion of AUH, an AU-specific RNA-binding enoyl-CoA hydratase. Gene 228, 85–91. 17 Nakagawa J & Moroni C (1997) A 20-amino-acid autonomous RNA-binding domain contained in an enoyl-CoA hydratase. Eur J Biochem 244, 890–899. 18 Kurimoto K, Fukai S, Nureki O, Muto Y & Yokoyama S (2001) Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-CoA hydratase. Structure (Cambridge) 9, 1253–1263. 19 Liesert M (2000) Untersuchungen Zur Biochemie der Vererbbaren Stoffwechselerkrankung Glutarazidurie Typ I, PhD Thesis, Philipps-Universita ¨ t, Marburg. 20 Ly TBN, Peters V, Gibson KM, Liesert M, Buckel W, Wilcken B, Carpenter K, Ensenauer R, Hoffmann GF, Mack M et al. (2003) Mutations in the AUH gene cause 3-methylglutaconic aciduria type I. Hum Mutat 21, 401– 407. M. Mack et al. Human 3-methylglutaconyl-CoA hydratase FEBS Journal 273 (2006) 2012–2022 ª 2006 The Authors Journal compilation ª 2006 FEBS 2021 [...].. .Human 3-methylglutaconyl-CoA hydratase M Mack et al 21 Buckel W, Dorn U & Semmler R (1981) Glutaconate CoA-transferase from Acidaminococcus fermentans Eur J Biochem 118, 315–321 22 Mack M, Bendrat K, Zelder O, Eckel E, Linder D & Buckel W (1994) Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans... 41–51 23 Martins BM, Dobbek H, Cinkaya I, Buckel W & Messerschmidt A (2004) Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin Proc Natl Acad Sci USA 101, 15645–15649 2022 24 Mack M & Buckel W (1995) Identification of glutamate beta 54 as the covalent-catalytic residue in the active site of glutaconate CoA-transferase from Acidaminococcus fermentans... Ziegert K & Eggerer H (1973) Acetyl-CoAdependent cleavage of citrate on inactivated citrate lyase Eur J Biochem 37, 295–304 26 Loupatty FJ, Ruiter JPLIJ, Duran M & Wanders RJ (2004) Direct nonisotopic assay of 3-methylglutaconylCoA hydratase in cultured human skin fibroblasts to specifically identify patients with 3-methylglutaconic aciduria type I Clin Chem 50, 1447–1450 FEBS Journal 273 (2006) 2012–2022 . Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism Matthias Mack 1 ,. homolog of enoyl-CoA hydratase ). The RNA- binding activity of the human protein and also of the murine homologue was investigated further, its biologi- cal