title: Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase.
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.
authors.affiliation: Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of Leon, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Malumbres
authors.forename: L M
authors.initials: LM
authors.lastname: Mateos
authors.forename: M A
authors.initials: MA
authors.lastname: Lumbreras
authors.forename: C
authors.initials: C
authors.lastname: Guerrero
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics*; D019757:Carbon-Oxygen Lyases; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C023160:threonine synthase