Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase.
AUTOR(ES)
Malumbres, M
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=201634Documentos Relacionados
- Nucleotide sequence of the threonine synthase (thrC) gene of Brevibacterium lactofermentum.
- Nucleotide sequence of the yeast THR4 gene encoding threonine synthase.
- Nucleotide sequence of the homoserine dehydrogenase (thr A) gene of Brevibacterium lactofermentum.
- Nucleotide sequence of the homoserine kinase (thr B) gene of Brevibacterium lactofermentum.
- Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12.