Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions.
Escherichia coli contains two L-asparaginase isozymes: a secreted high-affinity enzyme, L-asparaginase II (AnsII), and a low-affinity cytoplasmic enzyme, L-asparaginase I (AnsI), which is encoded by the ansA gene. The nucleotide sequence of ansA and flanking regions, comprising 2156 bp, has been determined. The ansA gene product has been identified and has a calculated Mr of 35,388; gel filtration of cell extracts indicates that the active form of the enzyme is a dimer. The deduced amino acid sequence of AnsI shows discernible similarity to AnsII in a region immediately adjacent to the proposed active-site peptide of asparaginase II as previously determined by substrate analogue binding experiments. A second open reading frame (ORF1), encoding a protein of Mr 23,336, is found 10 bp downstream from ansA; the ribosome-binding site of ORF1 overlaps the stop codon of ansA. Deletions within the 5' region of ansA abolish expression of ansA and also reduce expression of ORF1. Together, these observations suggest that ansA and ORF1 constitute an operon. A palindromic sequence exists in the 3' region of ORF1 which may function as a bidirectional transcription terminator both for the ansA-ORF1 operon and a second, convergent, ORF.
Jerlström
P G
PG
Division of Science and Technology, Griffith University, Nathan, Brisbane, Old, Australia.
Bezjak
D A
DA
Jennings
M P
MP
Beacham
I R
IR
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D007527:Isoenzymes; D001215:Asparaginase