Garcia Assuero F, Dyszy Fabio, Munte Claudia E, Demarco Ricardo, Beltramini Leila M, Oliva Glaucius, Costa-Filho Antonio J, Araujo Ana P U
Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil; Laboratório de Biofísica Molecular, Departamento de Física, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Biochim Biophys Acta. 2014 Jun;1844(6):1094-103. doi: 10.1016/j.bbapap.2014.03.005. Epub 2014 Mar 14.
In eukaryotes, there are still steps of the vitamin B1 biosynthetic pathway not completely understood. In Arabidopsis thaliana, THI1 protein has been associated with the synthesis of the thiazole ring, a finding supported by the identification of a thiamine pyrophosphate (TPP)-like compound in its structure. Here, we investigated THI1 and its mutant THI1(A140V), responsible for the thiamin auxotrophy in a A. thaliana mutant line, aiming to clarify the impact of this mutation in the stability and activity of THI1. Recently, the THI1 orthologue (THI4) was revealed to be responsible for the donation of the sulfur atom from a cysteine residue to the thiazole ring in the thiamine intermediate. In this context, we carried out a cysteine quantification in THI1 and THI1(A140V) using electron spin resonance (ESR). These data showed that THI1(A140V) contains more sulfur-containing cysteines than THI1, indicating that the function as a sulfur donor is conserved, but the rate of donation reaction is somehow affected. Also, the bound compounds were isolated from both proteins and are present in different amounts in each protein. Unfolding studies presented differences in melting temperatures and also in the concentration of guanidine at which half of the protein unfolds, thus showing that THI1(A140V) has its conformational stability affected by the mutation. Hence, despite keeping its function in the early steps during the synthesis of TPP precursor, our studies have shown a decrease in the THI1(A140V) stability, which might be slowing down the biological activity of the mutant, and thus contributing to thiamin auxotrophy.
在真核生物中，维生素B1生物合成途径的某些步骤仍未完全明确。在拟南芥中，THI1蛋白与噻唑环的合成有关，其结构中硫胺素焦磷酸（TPP）样化合物的鉴定支持了这一发现。在此，我们研究了导致拟南芥突变体系中硫胺素营养缺陷的THI1及其突变体THI1（A140V），旨在阐明该突变对THI1稳定性和活性的影响。最近，THI1的直系同源物（THI4）被发现负责将硫原子从半胱氨酸残基转移至硫胺素中间体的噻唑环。在此背景下，我们利用电子自旋共振（ESR）对THI1和THI1（A140V）中的半胱氨酸进行了定量分析。这些数据表明，THI1（A140V）含有的含硫半胱氨酸比THI1更多，这表明作为硫供体的功能得以保留，但供体反应速率受到了一定影响。此外，从两种蛋白质中分离出的结合化合物，其在每种蛋白质中的含量不同。展开研究显示，两种蛋白质在解链温度以及蛋白质解链一半时的胍浓度方面存在差异，从而表明THI1（A140V）的构象稳定性受到了该突变的影响。因此，尽管THI1（A140V）在TPP前体合成的早期步骤中仍保留其功能，但我们的研究表明其稳定性有所下降，这可能会减缓突变体的生物活性，进而导致硫胺素营养缺陷。
