Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.1.1.4 (leucyl-tRNA synthetase)
 297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.
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PMID:The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity. 1052 76

The 3.2 kb gene (leuS) encoding for leucyl-tRNA synthetase (LeuRS) has been cloned from E.coli K-12, and overexpressed 35 times more than that in the host strain TG1. In order to further increase the production of LeuRS, two types of leuS with different length of the 3' flanking region: leuS1 has an additional 130 bp over that of leuS2 were ligated into pKK-233-2 and pTrc-99B, respectively. In E. coli TG1 transformant harboring the recombination plasmid pTrc-99B with leuS2, the yield of LeuRS was 135 times that in TG1 strain. The purified enzyme that showed one band on SDS-PAGE was obtained after two steps of column chromatography. During the construction of plasmid, a substitute of G for C was introduced at position of base 4 in the coding region of leuS, so that Gln2 of LeuRS was changed to Glu. This enzyme was designated LeuRS2E. The kinetic constants of LeuRS2E showed that the substitution has no effect on the enzyme activity and could be used in the studies of LeuRS as the native enzyme.
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PMID:The Overproduction of Leucyl-tRNA Synthetase in E. coli and Its Purification. 1221 74