Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase

Abstract

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.Vanillate demethylase is a two-component enzyme classified as a IA oxygenase (25, 28). It comprises a reductase containing both a flavin and a [2Fe-2S] redox center and an oxygenase containing, in addition to a substrate-binding site, an ironbinding site and a Rieske-type [2Fe-2S] cluster. Little is known about how structure influences function in vanillate demethylase. Demethylases involved in the metabolism of p-anisate in Pseudomonas putida (1) and vanillate in P. testosteroni (3,35) and P. fluorescens (5) are known to be air sensitive and unstable. The vanillate demethylases from P. testosteroni and P. fluorescens are mixed-function oxygenases and have a wide substrate specificity: m-anisate, p-anisate, m-toluate, 3,4,5-trimethoxybenzoate, and 3,4-dimethoxybenzoate were oxidized by vanillate-induced cells (5, 36). As described here, the Acinetobacter vanillate demethylase also possesses a broad substrate range.Inferences can be drawn about the mechanism of vanillate demethylase from results obtained with the evolutionarily related enzyme phthalate dioxygenase (6). In this enzyme, electrons for hydroxylation flow from NADH to flavin mononucleotide to [2Fe-2S] in the reductase and from the Rieske-type [2Fe-2S] center to the Fe 2ϩ site in the oxygenase, where oxygen binding and hydroxylation occur (9,10,33,40). As recently shown for the naphthalene dioxygenase, another member of this group of aromatic dioxygenases, Fe1 of the Rieske [2Fe-2S] center is coordinated by two cysteinyl residues and Fe2 is coordinated by two histidyl residues (14,15,18). The iron atom at the active site is coord...

Expression and Substrate Range of <i>Streptomyces</i> Vanillate Demethylase

“…5,7,9) In a preliminary experiment, when a lysate was prepared from Streptomyces sp. NL15-2K cells exhibiting vanillate demethylase activity, activity of the enzyme was not detected in the lysate regardless of supplementation with cofactors such as reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH).…”

“…have been determined in studies using cell-free extracts or recombinant Escherichia coli cells transformed with the vanillate demethylase gene. [7][8][9] Vanillate demethylase from P. testosteroni has a broad substrate range and shows activity towards m-and p-anisate in addition to vanillate, 7) whereas the enzyme from P. fluorescens is inactive toward p-anisate. 8) Similarly, although vanillate demethylase from Acinetobacter sp.…”

Conversations with a Psychiatrist

“…It might be concluded that an objective analysis of how structure influences function is achieved more readily by confessions of ignorance guiding investigations using random mutagenesis than by assumptions of knowledge determining targets for site-directed mutagenesis. Particularly gratifying was success in the identification of essential residues in vanillate demethylase, a membrane-associated enzyme that has resisted purification (25). Combination of A. baylyi natural transformation with PCR mutagenesis and appropriate selection also made it possible to select strains that had altered binding targets for repressors (21) and altered substrate specificity for an enzyme (6).…”

When Coupled to Natural Transformation inAcinetobactersp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair

“…strain ADP1. These investigations have given valuable insight into structure-function relationships in these proteins (7,15,16,17,24,29). However, when we applied the random PCR mutagenesis approach to pcaK, a gene encoding a protocatechuate transporter belonging to the ubiquitous major facilitator superfamily (28), evidence of bias in the mutant collection was observed.…”

“…A merit of the procedure is its objectivity because mutations are identified on the basis of phenotypic variation. No presumptions are brought to bear on the identification of targets for mutagenesis, so amino acid contributions that might not have been predicted can be characterized (7,10,24). Nevertheless, the procedure must be regarded with caution because not all nucleotide substitutions are made with equal probability during PCR amplification (34).…”