Laboratório de Glicobiologia Estrutural e Funcional, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.
Laboratório de Glicobiologia Estrutural e Funcional, Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil; Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, UFRJ, Rio de Janeiro, RJ, Brazil.
J Biol Chem. 2021 Jan-Jun;296:100180. doi: 10.1074/jbc.RA120.015189. Epub 2020 Dec 17.
Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.
糖缀合物在许多细胞过程中起着核心作用，其组成的改变与许多人类病理学有关。细胞糖基化的底物是在己糖胺生物合成途径中合成的，该途径受谷氨酰胺：果糖-6-磷酸酰胺转移酶（GFAT）控制。人类同工型 2 GFAT（hGFAT2）与糖尿病和癌症有关；然而，关于这种酶的结构和酶学特性尚无信息。在这里，我们报告了一种成功的表达和纯化具有催化活性的重组 hGFAT2（rhGFAT2）的方法，该酶在大肠杆菌细胞中融合或不融合 C 末端的 HisTag。我们的酶动力学数据表明，hGFAT2 不遵循预期的有序双酶机制，并且 GlcN-6-P 合成速度比其他 GFAT 报道的要慢得多。此外，hGFAT2 能够在等摩尔量的谷氨酰胺存在下将果糖-6-磷酸异构化为葡萄糖-6-磷酸，从而导致无效的谷氨酰胺水解。rhGFAT2 的三维模型的结构分析，结合圆二色性数据，表明在谷氨酰胺酶结构域中存在一个部分结构的环，该环存在于真核酶中，但在大肠杆菌同源物中不存在。分子动力学模拟表明，该环是蛋白质中最灵活的部分，在 hGFAT2 的构象状态中起着关键作用。因此，我们的研究提供了 hGFAT2 的结构、动力学和力学的第一套全面数据，这无疑将有助于进一步研究 hGFAT2 的（病理）生理学。
