PMID:11440130
| Citation |
Ogawa-Miyata, Y, Kojima, H and Sano, K (2001) Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production. Biosci. Biotechnol. Biochem. 65:1149-54 |
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| Abstract |
Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine. |
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| Keywords |
Amino Acid Sequence; Aspartate Kinase/antagonists & inhibitors; Aspartate Kinase/chemistry; Aspartate Kinase/genetics; Aspartate Kinase/metabolism; Base Sequence; DNA Mutational Analysis; DNA Primers; Enzyme Stability; Escherichia coli/enzymology; Feedback; Hot Temperature; Molecular Sequence Data; Threonine/biosynthesis |
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