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PMID:8407838

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Citation

Fisher, KE and Eisenstein, E (1993) An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli. J. Bacteriol. 175:6605-13

Abstract

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.

Links

PubMed PMC206772

Keywords

Binding Sites; Chromosome Mapping; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Isoleucine/metabolism; Kinetics; Mutagenesis; Restriction Mapping; Structure-Activity Relationship; Threonine Dehydratase/genetics; Threonine Dehydratase/metabolism; Valine/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:ILVA

acts_upstream_of_or_within

GO:0009097: isoleucine biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:ILVA

enables

GO:0004794: L-threonine ammonia-lyase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:THD1

involved_in

GO:0009097: isoleucine biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:THD1

enables

GO:0004794: L-threonine ammonia-lyase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

See also

References

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