Laboratory of Structural Molecular Biology, Biosciences Institute, UNESP - São Paulo State University, São Vicente, SP, Brazil.
Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, Department of Biochemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil.
Appl Microbiol Biotechnol. 2020 Jun;104(12):5477-5492. doi: 10.1007/s00253-020-10491-5. Epub 2020 Apr 19.
The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys and Cys, respectively), and structural analysis showed that GLP and GLP were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.
韧皮部坏死菌 Xylella fastidiosa 属于黄单胞菌科，这是一个包含许多革兰氏阴性细菌的大家族，这些细菌会导致许多经济上重要作物的疾病。在该科不同属的基因组中发现了一个被注释为谷氧还蛋白样蛋白 (glp) 的预测基因，该基因高度保守且在 X. fastidiosa 中高度表达。对 GLP 蛋白序列的分析揭示了三个蛋白结构域：一个类似于单硫醇谷氧还蛋白 (Grx)，一个 Fe-S 簇和一个硫代硫酸盐硫转移酶/ 硫氰酸酶结构域 (Tst/Rho)，通常涉及硫代谢和氰化物解毒。为了表征 GLP 的生化特性，我们表达和纯化了 X. fastidiosa 重组 GLP 酶。未观察到 Grx 活性和 Fe-S 簇形成，而 Tst/Rho 酶活性评估表明 GLP 可以在体外解毒氰化物并将无机硫转移到受体分子。GLP 的生物学活性依赖于 Grx 和 Tst/Rho 结构域中的半胱氨酸残基（分别为 Cys 和 Cys），结构分析表明 GLP 和 GLP 能够形成高分子量寡聚物（>600 kDa），而 Cys 突变为 Ser 会使四级结构不稳定。在大肠杆菌中的异源酶表达实验表明，GLP 可以保护细菌免受高浓度氰化物和过氧化氢的侵害。最后，系统发育分析表明，同源 glp 基因分布在革兰氏阴性细菌科中，N 到 C 结构域的顺序保守。然而，没有真核生物含有这种酶。总之，这些结果表明 GLP 是一种重要的酶，具有细菌中的氰化物分解和硫转移酶功能，而真核生物中不存在这种酶，这代表了一种有前途的新型药物的生物靶标。
