Research Group Inborn Errors of Metabolism, Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany.
Research Group Inborn Errors of Metabolism, Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany; Institute for Functional Gene Analytics (IFGA), Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany.
Biochimie. 2021 Apr;183:30-34. doi: 10.1016/j.biochi.2021.02.002. Epub 2021 Feb 7.
Glycine conjugation is an important phase II reaction and represents a central detoxification pathway which is essential for the recycling of free coenzyme A. Only few sequence variants have been reported in the human GLYAT gene and only two studies have overexpressed the human protein in bacterial systems and partially characterized it. This has prompted us to study the wild-type enzyme and two sequence variants not only in the E. coli strain Origami 2(DE3), but also to overexpress GLYAT in HEK293 cells, a human-derived cell line. Following purification of the recombinant proteins from E. coli the wild-type GLYAT protein and sequence variants, p.(Gln61Leu) yielded decreased specific activity than the wild-type enzyme, while specific activity of p.(Asn156Ser) activity of the latter variant was somewhat increased. K values were similar for the three forms of GLYAT overexpressed in bacteria and for the wild-type enzyme overexpressed in HEK293 cells. Localization studies demonstrated intramitochondrial localization of human wild-type GLYAT, conjugated with eGFP, in the HEK293 cells. As p.(Gln61Leu) does not only impair GLYAT activity in vitro, but is of high prevalence in a Caucasian Afrikaner cohort in South Africa, potential pharmacogenetic implications, warrant further studies of GLYAT.
甘氨酸缀合是一种重要的 II 相反应，代表了一种重要的解毒途径，对于辅酶 A 的循环利用至关重要。人类 GLYAT 基因中仅报道了少数序列变异体，并且仅有两项研究在细菌系统中过表达了人类蛋白并对其进行了部分表征。这促使我们不仅在大肠杆菌 Origami 2(DE3)菌株中研究野生型酶和两种序列变异体，还在人源细胞系 HEK293 细胞中过表达 GLYAT。从大肠杆菌中纯化重组蛋白后，野生型 GLYAT 蛋白和序列变异体 p.(Gln61Leu)的比活性均低于野生型酶，而后者变异体 p.(Asn156Ser)的比活性略有增加。在细菌中过表达的三种形式的 GLYAT 和在 HEK293 细胞中过表达的野生型酶的 K 值相似。定位研究表明，在 HEK293 细胞中，与 eGFP 缀合的人野生型 GLYAT 定位于线粒体内部。由于 p.(Gln61Leu)不仅在体外降低了 GLYAT 的活性，而且在南非的一个白种阿非利卡人队列中也具有较高的普遍性，因此具有潜在的药物遗传学意义，需要进一步研究 GLYAT。
