ABSTRACT

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m⁷G) in tRNA. In tRNAs m⁷G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant K_d of TrmB and tRNA^Phe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNA^Phe complex structure.

摘要

TrmB 属于 I 类 S-腺苷甲硫氨酸 (SAM) 依赖性甲基转移酶 (MTase)，在 tRNA的第 7 位 (m ⁷ G)处将甲基引入鸟嘌呤。在 tRNA 中，m ⁷ G 最常见于可变环的第 46 位，并与 C13 和 U22 形成第三碱基对，在 G46 处引入正电荷。TrmB/Trm8 酶家族结构多样，因为 TrmB 蛋白以单体、同源二聚体和异源二聚体形式存在。到目前为止，确切的酶促机制以及 tRNA-TrmB 晶体结构尚不清楚。在这里，我们展示了枯草芽孢杆菌的第一个晶体结构TrmB 与 SAM 和 SAH 复合。TrmB apo 和与 SAM 和 SAH 复合的晶体结构已通过 X 射线晶体学确定为 1.9 Å (apo)、2.5 Å (SAM) 和 3.1 Å (SAH)。获得的晶体结构表明 Tyr193 在 SAM 结合和 MTase 活性期间很重要。应用荧光偏振，TrmB 和 tRNA ^{Phe的解离常数 K} _d被确定为 0.12 µM ± 0.002 µM。基于发光的甲基转移酶活性测定揭示了 TrmB 催化过程中的协同效应，在生理 SAM 浓度下具有一半的位点反应性。从小角 X 射线散射 (SAXS)、交联复合物的质谱和分子对接实验中检索到的结构数据导致了 TrmB-tRNA 的测定^复杂的结构。