Mack Matthias, Schniegler-Mattox Ute, Peters Verena, Hoffmann Georg F, Liesert Michael, Buckel Wolfgang, Zschocke Johannes
Institut für Technische Mikrobiologie der Hochschule Mannheim, Germany.
FEBS J. 2006 May;273(9):2012-22. doi: 10.1111/j.1742-4658.2006.05218.x.
The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized 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 mutations 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) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 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.
I型3-甲基戊二酸尿症（MGA1）是一种代谢性疾病，其特征在于有机酸谱异常，其中3-甲基戊二酸、3-甲基戊酸和3-羟基异戊酸的尿排泄过多。受影响个体表现出从轻度语言发育迟缓到伴有神经功能障碍的严重精神运动发育迟缓等多种临床表现。MGA1是由亮氨酸降解途径中3-甲基戊二酰辅酶A（3-MG-CoA）水合酶活性降低或缺失引起的。据报道，人类AUH基因编码一种具有RNA结合和烯酰辅酶A水合酶活性的双功能酶。此外，研究表明AUH基因的突变与MGA1有关。在此，我们展示了使用不同辅酶A底物时纯化的AUH基因产物的动力学数据。最佳底物是（E）-3-MG-CoA（Vmax = 3.9 U.mg-1，Km = 8.3 μM，kcat = 5.1 s-1）和（E）-戊二酰辅酶A（Vmax = 1.1 U.mg-1，Km = 2.4 μM，kcat = 1.4 s-1），这有力地证明了AUH基因在亮氨酸降解中编码主要的人类3-MG-CoA水合酶。基于这些结果，开发了一种用于成纤维细胞匀浆中AUH活性的新检测方法。在MGA1表型中发现的唯一错义突变c.719C>T，导致氨基酸交换A240V，产生的酶仅具有野生型3-MG-CoA水合酶活性的9%。
