Tài liệu Báo cáo khoa học: Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism docx

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:
(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.

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