Substrate range and genetic analysis of Acinetobacter vanillate demethylase.
An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.
Morawski
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Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Segura
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Ornston
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D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013696:Temperature; D014641:Vanillic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; D010090:Oxidoreductases, O-Demethylating; C023284:vanillate O-demethylase; D019729:Carbon-Oxygen Ligases; D014641:Vanillic Acid