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Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase

Morawski, Birgit and Segura, Ana and Ornston, L. Nicholas (2000) Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase. Journal of Bacteriology, 182 (5). pp. 1383-1389. ISSN 0021-9193.

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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.

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Segura, Ana0000-0002-2240-2452
Additional Information:Copyright © 2000, American Society for Microbiology. Received 30 August 1999/Accepted 14 December 1999 This research was supported by grants DAAG55-98-1-0232 from the Army Research Office and MCB-9603980 from the National Science Foundation. B.M. was supported by a postdoctoral fellowship from the Deutscher Akademischer Austauschdienst (DAAD). A.S. was supported by a postdoctoral fellowship from the Spanish Ministerio de Educacion y Ciencia. Publication 23 from the Biological Transformation Center in the Yale Biospherics Intitute.
Issue or Number:5
Record Number:CaltechAUTHORS:MORjbact00
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1854
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Deposited On:20 Feb 2006
Last Modified:09 Mar 2020 13:19

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