A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing.
A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing.
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DOI：
10.1021/bi062078j
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发表时间：
2007-05
期刊：
影响因子：
2.9
通讯作者：
M. Vu;S. Martinis
中科院分区：
文献类型：
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作者：
M. Vu;S. Martinis
亮氨酰-tRNA合成酶（LeuRS）是一种I类酶，其将其氨酰化活性位点容纳在由Rossmann核苷酸结合折叠定义的典型核心中。此外，许多LeuRS携带独特的多肽插入物，其由位于保守的KMSKS序列上游的约50个氨基酸组成。这种亮氨酸特异性结构域（LS结构域）的作用仍然不确定。我们假设，这个域可能是重要的底物识别在氨酰化和/或氨基酸编辑。我们进行了一系列的缺失突变和嵌合交换内的亮氨酸特异性结构域的大肠杆菌。我们的研究结果支持亮氨酸特异性结构域对氨酰化至关重要，但不是编辑活性所必需的。动力学分析确定LS结构域的缺失主要影响kcat。由于其接近的氨酰化活性位点，我们建议，这个域与tRNA在氨基酸活化和/或tRNA氨酰化过程中相互作用。虽然亮氨酸特异性结构域似乎对编辑复合物并不重要，但它仍然可能有助于将tRNA从氨酰化转移到编辑复合物的动态易位过程。
Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucine-specific domain is critical for aminoacylation but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts kcat. Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.