The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2′-PO₄ generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2′-PO₄ attacks NAD⁺ to form an RNA-2′-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential antifungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1Δ mutation and in vitro in NAD⁺-dependent conversion of a 2′-PO₄ to a 2′-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD⁺, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2′-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2′-PO₄ nucleotide with arabinose-2′-PO₄ diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2′-phospho-ADP-ribosylated intermediate. Our results implicate the 2′-PO₄ ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

中文翻译：

---

念珠菌、曲霉和球孢子菌 Tpt1 的活性和底物特异性：必需的 tRNA 剪接酶和潜在的抗真菌靶点


Tpt1 酶是真菌 tRNA 剪接的重要试剂，可去除真菌 tRNA 连接酶产生的内部 RNA 2'-PO ₄ 。 Tpt1 执行两步反应，其中： (i) 2'-PO ₄攻击 NAD ⁺形成 RNA-2'-磷酸-(ADP-核糖) 中间体； (ii) ADP-核糖 O2" 与 RNA 2'-磷酸二酯的酯交换产生 2'-OH RNA 和 ADP-核糖-1",2"-环状磷酸。由于Tpt1不参与后生动物tRNA剪接，且Tpt1敲除对哺乳动物生理没有明显影响，因此Tpt1被认为是潜在的抗真菌药物靶点。在这里，我们表征了来自四种人类真菌病原体的 Tpt1 酶：粗球孢子菌（Coccidioides immitis） ，谷热病的病原体；烟曲霉和白色念珠菌，在免疫功能低下的宿主中引起侵袭性、通常致命的感染；耳念珠菌（Candida auris） ，一种对当前疗法具有抗药性的新兴病原体。所有四种病原体 Tpt1 在体内均具有活性，可补充致命的酿酒酵母 tpt1 Δ 突变，并且在体外可将 2'-PO ₄转化为 2'-OH，依赖 NAD ⁺进行转化。真菌Tpt1s利用烟酰胺次黄嘌呤二核苷酸作为底物代替NAD ⁺ ，尽管亲和力低得多，而烟酸腺嘌呤二核苷酸则无效。真菌 Tpt1s 有效地从全 DNA 底物中去除了内部核糖核苷酸 2'-磷酸。 用阿拉伯糖-2'-PO ₄替换 RNA 核糖-2'-PO ₄核苷酸可使酶比活性降低 ≥2000 倍，并选择性地减慢反应途径的步骤 2，导致 ara-2'- 的瞬时积累磷酸-ADP-核糖基化中间体。我们的结果表明 2'-PO ₄核糖核苷酸是真菌 Tpt1 核酸底物特异性的主要决定因素。