Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Reno, NV, USA.
FEBS J. 2022 May;289(10):2948-2958. doi: 10.1111/febs.16316. Epub 2021 Dec 13.
3-Methylglutaconic (3MGC) aciduria occurs in numerous inborn errors associated with compromised mitochondrial energy metabolism. In these disorders, 3MGC CoA is produced de novo from acetyl CoA in three steps with the final reaction catalysed by 3MGC CoA hydratase (AUH). In in vitro assays, whereas recombinant AUH dehydrated 3-hydroxy-3-methylglutaryl (HMG) CoA to 3MGC CoA, free CoA was also produced. Although HMG CoA is known to undergo non-enzymatic intramolecular cyclisation, forming HMG anhydride and free CoA, the amount of free CoA generated increased when AUH was present. To test the hypothesis that the AUH-dependent increase in CoA production is caused by intramolecular cyclisation of 3MGC CoA, gas chromatography-mass spectrometry analysis of organic acids was performed. In the absence of AUH, HMG CoA was converted to HMG acid while, in the presence of AUH, 3MGC acid was also detected. To determine which 3MGC acid diastereomer was formed, immunoblot assays were conducted with 3MGCylated BSA. In competition experiments, when α-3MGC IgG was preincubated with trans-3MGC acid or cis-3MGC acid, the cis diastereomer inhibited antibody binding to 3MGCylated BSA. When an AUH assay product mix served as competitor, α-3MGC IgG binding to 3MGCylated BSA was also inhibited, indicating cis-3MGC acid is produced in incubations of AUH and HMG CoA. Thus, non-enzymatic isomerisation of trans-3MGC CoA drives AUH-dependent HMG CoA dehydration and explains the occurrence of cis-3MGC acid in urine of subjects with 3MGC aciduria. Furthermore, the ability of cis-3MGC anhydride to non-enzymatically acylate protein substrates may have deleterious pathophysiological consequences.
3-甲基戊烯二酸（3MGC）酸尿症发生在许多与线粒体能量代谢受损有关的先天性错误中。在这些疾病中，3MGC CoA 由乙酰 CoA 在三步中从头产生，最后一步由 3MGC CoA 水合酶（AUH）催化。在体外测定中，虽然重组 AUH 将 3-羟基-3-甲基戊二酰辅酶 A（HMG CoA）脱水为 3MGC CoA，但也产生了游离 CoA。尽管已知 HMG CoA 会经历非酶促分子内环化，形成 HMG 酐和游离 CoA，但当存在 AUH 时，生成的游离 CoA 量增加。为了测试 AUH 依赖性 CoA 生成增加是由 3MGC CoA 的分子内环化引起的假设，对有机酸进行了气相色谱-质谱分析。在不存在 AUH 的情况下，HMG CoA 转化为 HMG 酸，而在存在 AUH 的情况下，也检测到 3MGC 酸。为了确定形成哪种 3MGC 酸差向异构体，用 3MGCylated BSA 进行了免疫印迹测定。在竞争实验中，当 α-3MGC IgG 与反式-3MGC 酸或顺式-3MGC 酸预孵育时，顺式差向异构体抑制抗体与 3MGCylated BSA 的结合。当 AUH 测定产物混合物作为竞争物时，α-3MGC IgG 与 3MGCylated BSA 的结合也受到抑制，表明在 AUH 和 HMG CoA 的孵育中产生了顺式-3MGC 酸。因此，反式 3MGC CoA 的非酶异构化驱动 AUH 依赖性 HMG CoA 脱水，并解释了 3MGC 酸尿症患者尿液中顺式 3MGC 酸的存在。此外，顺式 3MGC 酐非酶酰化蛋白底物的能力可能具有有害的病理生理后果。
