|
type |
Journal Article |
authors |
Malumbres, M.; Mateos, L. M.; Lumbreras, M. A.; Guerrero, C.; Martin, J. F. |
title |
Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase |
journal |
Appl Environ Microbiol |
Activity |
4.2.3.1 |
Family |
4.2.3.1 |
sel |
selected |
ui |
8074505 |
year |
(1994) |
volume |
60 |
number |
7 |
pages |
2209-19 |
| |
keywords |
Amino Acid Sequence |
abstract |
The thrC gene of Brevibacterium lactofermentum was cloned by complementation of Escherichia coli thrC auxotrophs. The gene was located by deletion mapping and complementation analysis in a 2.9-kb Sau3AI-HindIII fragment of the genome. This fragment also complemented a B. lactofermentum UL1035 threonine auxotroph that was deficient in threonine synthase. A 1,892-bp DNA fragment of this region was sequenced; this fragment contained a 1,446-bp open reading frame that encoded a 481-amino-acid protein having a deduced M(r) of 52,807. The gene was expressed in E. coli, by using the phage T7 system, as a 53- kDa protein. The promoter region subcloned in promoter-probe plasmids was functional in E. coli. A Northern analysis revealed that the gene was expressed as a monocistronic 1,400-nucleotide transcript. The transcription start point of the thrC gene was located by S1 mapping 6 bp upstream from the translation initiation codon, which indicated that this promoter was one of the leaderless transcription-initiating sequences. The threonine synthase overexpressed in B. lactofermentum UL1035 was purified almost to homogeneity. The active form corresponded to a monomeric 52.8-kDa protein, as shown by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The purified enzyme required pyridoxal phosphate as its only cofactor to convert homoserine phosphate into threonine. |
last changed |
2009/06/26 10:21 |
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