Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases.
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.
Zhao
Ming-Wei
MW
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Hao
Rui
R
Chen
Jian-Feng
JF
Martin
Franck
F
Eriani
Gilbert
G
Wang
En-Duo
ED
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D022761:Gene Transfer, Horizontal; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D012356:RNA, Transfer, Leu / Q000378:metabolism*; D013329:Structure-Activity Relationship
D012356:RNA, Transfer, Leu; D000604:Amino Acyl-tRNA Synthetases