丙胺酸-tRNA 合成酶 (Alanyl-tRNA synthetase, AlaRS) 屬於轉譯酵素中的一員，負責將丙胺酸 (Alanine) 轉移到其同源 tRNA上，形成 Ala-tRNAAla，然後將其遞送至核醣體為著蛋白質合成。AlaRS都有四個功能域，包含「催化結構域」、「tRNA 辨識結構域域」、「校正結構域」和「C-Ala結構域」。在AlaRS的tRNA 辨識結構域中有兩個高度保守的胺基酸殘基N及D (例如E. coli AlaRS的 N303 和 D400) 與 tRNAAla 中的接受柄上的G3:U70有專一性的交互作用。有趣的是，一種天然存在的mini-AlaRS被發現於卡氏棘阿米巴 (Acanthamoeba castellanii) 中的圖邦病毒 (Tupanvirus)，該酵素缺乏校正結構域和 C-Ala 結構域，並且在其 tRNA 辨識結構域中含有的是 P/T 而不是 N/D。親緣演化分析顯示，這種mini-AlaRS的親緣關係比起原核生物的AlaRS，與真核生物AlaRS的關係更緊密，體現出它起源於真核生物。胺醯化分析 (Aminoacylation assay) 顯示，儘管缺乏 N/D 胺基酸，但圖邦病毒的 AlaRS (TuAlaRS) 能夠專一地辨認含有G3:U70 的tRNAAla。然而奇怪的是，突變P/T卻對它的胺醯化活性或專一性沒有影響。另外，TuAlaRS能以相似的效率作用於microhelixAla；相對的，E. coli AlaRS偏好作用於tRNAAla過於microhelixAla。電泳遲緩分析更進一步顯示圖邦病毒AlaRS 與野生型 (G3:U70) 和突變型 (非 G3:U70) 的tRNAAla和microhelixAla 的結合有相似的親和度；而E. coli AlaRS偏向於與野生型tRNAAla結合。由於缺乏校正結構區域，因此圖邦病毒AlaRS相較於E. coli酵素，表現出更高機率的錯誤性胺醯化作用。本研究的重點在於這自然存在於mini-AlaRS的強力丙胺酸化活性。;Alanyl-tRNA synthetase (AlaRS) belongs to a family of translation enzymes and is responsible for transferring alanine to its cognate tRNA, forming Ala-tRNAAla, which is then delivered to ribosomes for protein synthesis. AlaRS contains four functional domains: catalysis, tRNA-recognition, editing, and C-Ala domains. Two highly conserved amino acid residues N and D in the tRNA-recognition domain of AlaRS (N303 and D400 in E. coli AlaRS) make specific contacts with the identity element G3:U70 in the acceptor stem of tRNAAla. Interestingly, a naturally occurring mini-AlaRS was identified in the Tupanvirus of Acanthamoeba castellanii. This enzyme lacks the editing and C-Ala domains and contains P/T instead of N/D in its tRNA-recognition domain. Phylogenetic analysis indicated that this mini-AlaRS is more closely related to eukaryotic AlaRSs than to prokaryotic AlaRSs, suggesting its eukaryotic origin. Aminoacylation assay showed that Tupanvirus AlaRS specifically charges G3:U70-containing tRNAAla despite lacking the N/D residues. However, mutation of P/T had little effect on its aminoacylation activity or specificity. In addition, this enzyme could charge microhelixAla to a similar level. In contrast, E. coli AlaRS strongly preferred tRNAAla over microhelixAla. Electrophoretic mobility shift assay further showed that Tupanvirus AlaRS bound wild-type (G3:U70) and mutant (non-G3:U70) tRNAAla and microhelixAla with a similar affinity, while E. coli AlaRS preferentially bound WT tRNAAla. Due to the lack of the editing domain, Tupanvirus AlaRS exhibited a higher misacylation rate compared with the E. coli enzyme. This study highlights the strong alanylation activity of a naturally occurring mini-AlaRS.
