Enzymes assembled from Aquifex aeolicus and Escherichia coli Leucyl-tRNA synthetases

	Enzymes assembled from Aquifex aeolicus and Escherichia coli Leucyl-tRNA synthetases
	Zhao, MW; Hao, R; Chen, JF; Martin, F; Eriani, G; Wang, ED
刊名	BIOCHEMISTRY
	2003
卷号	42 期号:25 页码:7694-7700
通讯作者	Wang, ED (reprint author), Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, State Key Lab Mol Biol, Shanghai 200031, Peoples R China.,
英文摘要	Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha-and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.
学科主题	Biochemistry & Molecular Biology
类目[WOS]	Biochemistry & Molecular Biology
关键词[WOS]	BINDING ; COMPLEX ; DISCRIMINATION ; OVERPRODUCTION ; PURIFICATION ; EXPRESSION ; TRNA(LEU) ; REVEALS ; GENE
收录类别	SCI
语种	英语
WOS记录号	WOS:000183821200007
内容类型	期刊论文
版本	出版稿
源URL	[http://202.127.25.143/handle/331003/2388]
专题	上海生化细胞研究所_上海生科院生化细胞研究所
推荐引用方式 GB/T 7714	Zhao, MW,Hao, R,Chen, JF,et al. Enzymes assembled from Aquifex aeolicus and Escherichia coli Leucyl-tRNA synthetases[J]. BIOCHEMISTRY,2003,42(25):7694-7700.
APA	Zhao, MW,Hao, R,Chen, JF,Martin, F,Eriani, G,&Wang, ED.(2003).Enzymes assembled from Aquifex aeolicus and Escherichia coli Leucyl-tRNA synthetases.BIOCHEMISTRY,42(25),7694-7700.
MLA	Zhao, MW,et al."Enzymes assembled from Aquifex aeolicus and Escherichia coli Leucyl-tRNA synthetases".BIOCHEMISTRY 42.25(2003):7694-7700.