The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.
The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.
复制标题
糖基天冬酰胺酶的 T99K 变体显示了遗传病天冬氨葡萄糖胺尿症的新结构机制。
DOI：
10.1002/pro.3607
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Guo,Hwai-Chen
中科院分区：
文献类型：
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作者：
Pande,Suchita;Guo,Hwai-Chen
天冬氨酸氨基葡萄糖尿症(AGU)是一种遗传性疾病，由一种名为天冬氨酸氨基葡萄糖苷酶(AGA)或糖基天冬酰胺酶(GA)的溶酶体酰胺酶突变引起。这种疾病导致糖天冬酰胺在几乎所有细胞类型的溶酶体中积聚，并伴有严重的临床症状，影响中枢神经系统、骨骼异常和结缔组织病变。GA是作为单链前体合成的，需要分子内自动处理才能形成成熟的酰胺酶。此前，我们表明，加拿大的AGU突变破坏了这一强制性的分子内自动加工，使酶成为不活跃的前体。在这里，我们报告了一种与美国新的AGU等位基因T99K变种相对应的模型酶的生化和结构特征。与其他已知3D结构的变体不同，这种T99K模型酶仍然具有自动处理能力，可以生成成熟的形式。然而，它消化糖天冬酰胺的酰胺酶活性仍然很低，这与它与AGU的联系是一致的。我们确定了这种新的AGU模型酶的1.5？分辨率结构，并构建了酶-底物复合体，为分析T99K点突变对酰胺酶KMandkcat的负面影响提供了结构基础。看来，一种能够修复二聚体界面局部紊乱的“分子钳”可能能够弥补这种新的AGU变体的缺陷。
Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single‐chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5‐Å‐resolution structure of this new AGU model enzyme and built an enzyme–substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation onKMandkcatof the amidase. It appears that a “molecular clamp” capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.