An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.
High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation.
Fisher
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KE
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, Rockville 20850.
Eisenstein
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D001665:Binding Sites; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D013913:Threonine Dehydratase / Q000235:genetics* / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D007532:Isoleucine; D013913:Threonine Dehydratase; D014633:Valine