Cloning and nucleotide sequence of the gene coding for aspartokinase II from a thermophilic methylotrophic Bacillus sp.
The structural gene coding for the lysine-sensitive aspartokinase II of the methylotrophic thermotolerant Bacillus sp. strain MGA3 was cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking all three aspartokinase isozymes. The nucleotide sequence of the entire 2.2-kb PstI fragment was determined, and a single open reading frame coding for the aspartokinase II enzyme was found. Aspartokinase II was shown to be an alpha 2 beta 2 tetramer (M(r) 122,000) with the beta subunit (M(r) 18,000) encoded within the alpha subunit (M(r) 45,000) in the samea reading frame. The enzyme was purified, and the N-terminal sequences of the alpha and beta subunits were identical with those predicted from the gene sequences. The predicted amino acid sequence was 76% identical with the sequence of the Bacillus subtilis aspartokinase II. The transcription initiation site was located approximately 350 bp upstream of the translation start site, and putative promoter regions at -10 (TATGCT) and -35 (ATGACA) were identified. A 300-nucleotide intervening sequence between the transcription initiation and translational start sites suggests a possible attenuation mechanism for the regulation of transcription of this enzyme in the presence of lysine.
Schendel
F J
FJ
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108.
Flickinger
M C
MC
D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D005798:Genes, Bacterial; D007438:Introns / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008239:Lysine / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003062:Codon; D007527:Isoenzymes; D010455:Peptides; D001222:Aspartate Kinase; D008239:Lysine