Koster Kira-Lee, Sturm Marga, Herebian Diran, Smits Sander H J, Spiekerkoetter Ute
Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children's Hospital, Moorenstr.5, 40225, Duesseldorf, Germany.
J Inherit Metab Dis. 2014 Nov;37(6):917-28. doi: 10.1007/s10545-014-9732-5. Epub 2014 Jun 26.
Medium-chain acyl-coenzyme-A dehydrogenase (MCAD) catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs. Mutations in the ACADM gene cause MCAD deficiency presenting with life-threatening symptoms during catabolism. Since fatty-acid-oxidation disorders are part of newborn screening (NBS), many novel mutations with unknown clinical relevance have been identified in asymptomatic newborns. Eighteen of these mutations were separately cloned into the human ACADM gene, heterologously overexpressed in Escherichia coli and functionally characterized by using different substrates, molecular chaperones, and measured at different temperatures. In addition, they were mapped to the three-dimensional MCAD structure, and cross-link experiments were performed. This study identified variants that only moderately affect the MCAD protein in vitro, such as Y42H, E18K, and R6H, in contrast to the remaining 15 mutants. These three mutants display residual octanoyl-CoA oxidation activities in the range of 22 % to 47 %, are as temperature sensitive as the wild type, and reach 100 % activity with molecular chaperone co-overexpression. Projection into the three-dimensional protein structure gave some indication as to possible reasons for decreased enzyme activities. Additionally, six of the eight novel mutations, functionally characterized for the first time, showed severely reduced residual activities < 5 % despite high expression levels. These studies are of relevance because they classify novel mutants in vitro on the basis of their corresponding functional effects. This basic knowledge should be taken into consideration for individual management after NBS.
中链酰基辅酶A脱氢酶（MCAD）催化中链酰基辅酶A线粒体β氧化的第一步。ACADM基因突变会导致MCAD缺乏，在分解代谢期间出现危及生命的症状。由于脂肪酸氧化障碍是新生儿筛查（NBS）的一部分，因此在无症状新生儿中已鉴定出许多临床相关性未知的新突变。这些突变中有18个分别克隆到人类ACADM基因中，在大肠杆菌中进行异源过表达，并通过使用不同底物、分子伴侣在不同温度下进行功能表征。此外，将它们映射到MCAD的三维结构上，并进行交联实验。与其余15个突变体相比，本研究鉴定出在体外仅对MCAD蛋白有中度影响的变体，如Y42H、E18K和R6H。这三个突变体的残留辛酰辅酶A氧化活性在22%至47%的范围内，与野生型一样对温度敏感，在分子伴侣共过表达时活性达到100%。投影到三维蛋白质结构中为酶活性降低的可能原因提供了一些线索。此外，首次进行功能表征的八个新突变中有六个，尽管表达水平很高，但残留活性严重降低<5%。这些研究具有相关性，因为它们根据相应的功能效应在体外对新突变体进行分类。在新生儿筛查后进行个体管理时应考虑这些基础知识。
