Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD
Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD
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DOI：
10.1261/rna.2234310
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发表时间：
2010-11-01
期刊：
影响因子：
4.5
通讯作者：
Remme, Jaanus
中科院分区：
文献类型：
--
作者：
Ero, Rya;Leppik, Margus;Remme, Jaanus
沿着核糖体组装途径，会发生各种核糖体 RNA 加工和修饰反应。大肠杆菌核糖体大亚基中的茎环 69 在核糖体功能中发挥着重要作用。它含有由假尿苷合酶 RluD 合成的三个高度保守的假尿苷。其中一个假尿苷被 RlmH 进一步甲基化。在本文中，我们证明 RlmH 在 rRNA 修饰酶中具有独特的底物特异性。它优先甲基化假尿苷，而较低效率地甲基化尿苷。此外，RlmH 是唯一已知的 70S 核糖体特异性修饰酶。 RlmH测定的动力学参数如下：底物70S核糖体的表观K-M为0.51+/-0.06μM，辅因子S-腺苷-L-甲硫氨酸为27+/-3μM； k(cat) 值分别为 4.95 +/- 1.10 min(-1) 和 6.4 +/- 1.3 min(-1)。了解 RlmH 的底物特异性和动力学参数也使得确定 RluD 的动力学参数成为可能。底物50S亚基的K-M值为0.98+/-0.18μM，k(cat)值为1.97+/-0.46min(-1)。 RluD 是第一个进行动力学表征的 rRNA 假尿苷合酶。确定的 23S rRNA 的 RluD 和 RlmH 定向修饰速率与体内 50S 组装的速率一致。 RlmH 需要 30S 亚基的事实表明 50S 亚基成熟依赖于 30S 亚基的同时存在。
Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K-M for substrate 70S ribosomes is 0.51 +/- 0.06 mu M, and for cofactor S-adenosyl-L-methionine 27 +/- 3 mu M; the k(cat) values are 4.95 +/- 1.10 min(-1) and 6.4 +/- 1.3 min(-1), respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K-M value for substrate 50S subunits is 0.98 +/- 0.18 mu M and the k(cat) value is 1.97 +/- 0.46 min(-1). RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.