Dobson C Melissa, Gradinger Abigail, Longo Nicola, Wu Xuchu, Leclerc Daniel, Lerner-Ellis Jordan, Lemieux Melissa, Belair Caroline, Watkins David, Rosenblatt David S, Gravel Roy A
Department of Biochemistry and Molecular Biology, University of Calgary, Canada.
Mol Genet Metab. 2006 Aug;88(4):327-33. doi: 10.1016/j.ymgme.2006.03.009. Epub 2006 May 11.
Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of D- and L-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient's fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C>T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41-160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of D- to L-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient's MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.
甲基丙二酰辅酶A差向异构酶（MCE）在支链氨基酸、奇数链长脂肪酸及其他代谢产物的降解途径中催化D-甲基丙二酰辅酶A和L-甲基丙二酰辅酶A的相互转化。尽管在MCE上游紧邻的酶促步骤（丙酰辅酶A羧化酶）和下游（腺苷钴胺素依赖性甲基丙二酰辅酶A变位酶）发生了代谢紊乱，但尚未发现影响MCE自身的致病突变。一名曾被鉴定为属于维生素B12紊乱的cblA互补组但在受影响基因MMAA中缺乏突变的患者，接受了MCEE基因的突变检测。与对照组相比，该患者的成纤维细胞中腺苷钴胺素水平正常，而其他cblA细胞系通常辅因子水平降低。此外，该患者的甲基丙二酸尿症形式比通常在cblA患者中观察到的要轻。通过序列分析发现该患者MCEE基因存在c.139C>T（p.R47X）突变的纯合子，PCR产物的限制性消化证实了这一点。一名同样患有轻度甲基丙二酸尿症的同胞也是该突变的纯合子。父母双方和另一名同胞是杂合子。一个附近的插入多态性位点c.41-160_161insT，父母双方均为杂合子，在突变等位基因上显示野生型构型。为了评估培养细胞中孤立的MCE缺乏的影响，用含有MCEE特异性小干扰RNA（siRNA）的可选择载体转染HeLa细胞以抑制基因表达。MCEE mRNA水平的降低导致[14C] - 丙酸盐掺入细胞大分子的减少。然而，siRNA仅导致途径活性略有降低，这表明先前推测的D-甲基丙二酰辅酶A向L-甲基丙二酰辅酶A的非酶促转化可能对该途径的一些通量有贡献。我们得出结论，患者的MCEE缺陷是导致轻度甲基丙二酸尿症的原因，证实了人类对该酶活性的部分需求。
