Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli.
A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome. Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase. DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine. The deduced amino acid sequence for the 47.2-kilodalton subunit of E. coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N- and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007522:Isocitrate Lyase / Q000235:genetics*; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000235:genetics*; D013329:Structure-Activity Relationship
D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase