Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase
Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase
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DOI：
10.1006/jmbi.1999.2708
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发表时间：
1999-05-14
影响因子：
5.6
通讯作者：
Spremulli, LL
中科院分区：
文献类型：
--
作者：
Bullard, JN;Cai, YC;Spremulli, LL
人类线粒体苯丙氨酰-tRNA合成酶(MtPheRS)已经从人类EST数据库中被鉴定出来。利用来自细菌phers的α和β亚基保守区的共同序列，鉴定了两个部分测序的cDNA克隆。出乎意料的是，序列分析表明，其中一个克隆是另一个克隆的截短形式。详细的分析表明，与原核和真核细胞质形式的PHERS的(α-β)(2)结构不同，人mtPheRS由单一的多肽链组成。该蛋白已被克隆并在大肠杆菌中表达。凝胶过滤和分析速度沉淀离心法表明，人mtPheRS以单体形式存在。N端的314个氨基酸残基似乎类似于原核生物Phers的α亚基，而C端的100个氨基酸残基对应于已知与tRNA(Phe)反密码子相互作用的β亚基区域。与酵母和果蝇线粒体phers序列的比较表明，它们分别与人类mtPheRS有42%和51%的同源性。序列分析证实了II类氨酰-tRNA合成酶的特征基序的存在。测定了ATP：PPI交换和人mtPheRS进行的氨酰化反应的K-M和k(CAT)值。这种人类mtPheRs小单体的进化起源尚不清楚，然而，暗示这种酶是更复杂的(α-β)(2)细菌phers简化的结果，其中特定的功能区被保留。(C)1999年学术出版社。
Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identified from the human EST database. Using consensus sequences derived from conserved regions of the alpha and beta-subunits from bacterial PheRS, two partially sequenced cDNA clones were identified. Unexpectedly, sequence analysis indicated that one of these clones was a truncated form of the other. Detailed analysis indicates that unlike the (alpha beta)(2) structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS consists of a single polypeptide chain. This protein has been cloned and expressed in Escherichia coli. Gel filtration and analytical velocity sedimentation centrifugation indicate that the human mtPheRS is active in a monomeric form. The N-terminal 314 amino acid residues appear to be analogous to the alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino acid residues correspond to a region of the beta-subunit known to interact with the anticodon of tRNA(Phe). Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42% and 51% identical with the human mtPheRS, respectively. Sequence analysis confirms the presence of motifs characteristic of class II aminoacyl-tRNA synthetases. K-M and k(cat) values for ATP:PPi exchange and for the aminoacylation reaction carried out by human mtPheRS have been determined. Evolutionary origins of this small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alpha beta)(2) bacterial PheRS in which specific functional regions were retained. (C) 1999 Academic Press.