Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2′,3′-cyclic-PO₄ and 5′-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3′-OH,2′-PO₄ and 5′-PO₄ termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and are promising targets for antifungal drug discovery because their domain compositions and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. A distinctive feature of Trl1 is its preferential use of GTP as phosphate donor for the RNA kinase reaction. Here we report the 2.2 Å crystal structure of the kinase domain of Trl1 from the fungal pathogen Candida albicans with GDP and Mg²⁺ in the active site. The P-loop phosphotransferase fold of the kinase is embellished by a unique ‘G-loop’ element that accounts for guanine nucleotide specificity. Mutations of amino acids that contact the guanine nucleobase efface kinase activity in vitro and Trl1 function in vivo. Our findings fortify the case for the Trl1 kinase as an antifungal target.

中文翻译：

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真菌tRNA连接酶的RNA激酶结构域GTP特异性的结构基础

真菌tRNA连接酶（Trl1）是一种必需的酶，可修复在真菌未折叠的蛋白质反应中在tRNA剪接和非经典mRNA剪接过程中造成的2'，3'-PO ₄和5'-OH末端的RNA断裂。Trl1由C端环状磷酸二酯酶和中央多核苷酸激酶结构域组成，该结构域修复了末端以产生3'-OH，2'-PO ₄和5'-PO ₄N末端连接酶结构域密封所需的末端。Trl1酶存在于所有人类真菌病原体中，并有望成为抗真菌药物发现的靶标，因为与哺乳动物RtcB型tRNA剪接酶相比，它们的结构域组成和生化机制是独特的。Trl1的一个显着特征是它优先使用GTP作为RNA激酶反应的磷酸盐供体。在这里，我们报道了来自真菌病原体白色念珠菌的Trl1激酶结构域的2.2Å晶体结构，其中活性位点具有GDP和Mg ²⁺。独特的“ G环”元件修饰了激酶的P环磷酸转移酶折叠，这说明了鸟嘌呤核苷酸的特异性。接触鸟嘌呤核苷表面激酶活性的氨基酸突变体外和Trl1体内功能。我们的发现加强了Trl1激酶作为抗真菌靶标的作用。