Tài liệu Báo cáo khoa học: Biochemical characterization of human 3-methylglutaconyl-CoA hydratase

Biochemical characterization of human

3-methylglutaconyl-CoA hydratase and its role in leucine

metabolism

Matthias Mack1

, Ute Schniegler-Mattox2

, Verena Peters3

, Georg F. Hoffmann3

, Michael Liesert4

,

Wolfgang Buckel4 and Johannes Zschocke2

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: [email protected]

(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 (Vmax ¼ 3.9 UÆmg)1

, Km ¼ 8.3 lm,

kcat ¼ 5.1 s)1

) and (E)-glutaconyl-CoA (Vmax ¼ 1.1 UÆmg)1

, Km ¼ 2.4 lm,

kcat 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