Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans
Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans
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DOI：
10.1016/s1096-7192(02)00152-x
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发表时间：
2002-09-01
影响因子：
3.8
通讯作者：
Vockley, J
中科院分区：
文献类型：
--
作者：
Nguyen, TV;Andresen, BS;Vockley, J
酰基辅酶A脱氢酶（acyl-CoA dish ases，ACD）是催化酰基辅酶A酯的α，β-脱氢的相关酶家族。先前已经鉴定了两种在支链氨基酸代谢中具有活性的同系物。我们已经使用大肠杆菌中的表达，以产生一个以前未表征的ACD样序列（ACAD 8），并定义其底物特异性。纯化的重组酶以异丁酰辅酶A、（S）2-甲基丁酰辅酶A和正丙酰辅酶A为底物时，k（cat）/K-m分别为0.8、0.23和0.04（μ M（-1）s（-1））。因此，这种酶是异丁酰辅酶A脱氢酶。先前已经描述了单个患者，其成纤维细胞表现出特定的缬氨酸氧化缺陷。从患者成纤维细胞mRNA制备的扩增的A CAD 8 cDNA对于A CAD 8编码区中的单核苷酸变化（905 G> A）是纯合的。与来自对照细胞的序列相比。这在全长蛋白质中编码Arg 302 Gln取代（在成熟蛋白质中的位置280），通过分子建模预测该位置在亚基相互作用中是重要的。该突变体酶在大肠杆菌中表达时稳定但无活性。它也是稳定的，并适当地靶向线粒体，但在哺乳动物细胞中表达时无活性。这些数据进一步证实了在人中存在对缬氨酸catalysts（异丁酰-CoA脱氢酶，IBDH）特异性的分离ACD，沿着首次酶促和分子学证实了患者中该酶的缺乏。(C)2002 Elsevier Science（美国）。All rights reserved.
The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity. Purified recombinant enzyme had a k(cat)/K-m of 0.8, 0.23, and 0.04 (muM(-1)s(-1)) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionyl-CoA, respectively, as substrates. Thus, this enzyme is an isobutyryl-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified A CAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G > A) in the A CAD8 coding re.-ion compared to the sequence from control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but inactive when expressed in E coli. It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient. (C) 2002 Elsevier Science (USA). All rights reserved.