Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a “heal and seal” pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a “direct ligation” pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.

中文翻译：

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毛霉目真菌表达“仅密封”tRNA 连接酶

一些真核生物前 tRNA 含有一个内含子，可被一组专用酶去除。含有内含子的前 tRNA 被 tRNA 剪接核酸内切酶切割，然后连接两个外显子并释放内含子。真菌使用“愈合和密封”途径，需要 tRNA 连接酶 Trl1 的三个不同催化结构域。相比之下，人类使用 RTCB 执行的“直接连接”途径，RTCB 是一种与 Trl1 完全无关的酶。由于这些机制上的差异，Trl1 被提议作为真菌感染的有希望的药物靶点。为了验证 Trl1 作为广谱药物靶点，我们证明来自三个不同门的真菌含有具有所有三个结构域的 Trl1 直向同源物。这包括主要的侵入性人类真菌病原体，并且这些蛋白质各自可以在功能上替代酵母 Trl1。相比之下，毛霉目物种，包括病原体无根根霉和卷枝毛霉，具有非典型的 Trl1，其中包含密封结构域，但缺乏两个愈合结构域。尽管这些物种比其他病原真菌含有更少的 tRNA 内含子，但它们仍然需要剪接来解码 61 个有义密码子中的三个。这些仅密封的 Trl1 直向同源物可以在功能上补充酵母 Trl1 相应结构域中的缺陷，并使用保守的催化赖氨酸残基。我们得出的结论是，毛霉目使用一种仅封闭的酶与未识别的非直系同源的愈合酶一起用于其愈合和封闭途径。这意味着针对封闭活性的药物比针对愈合域的药物更有可能是广谱抗真菌药。