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The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

ABSTRACT: In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.

SUBMITTER: Marelja Z

PROVIDER: S-EPMC4055135 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

Marelja Zvonimir Z Dambowsky Miriam M Bolis Marco M Georgiou Marina L ML Garattini Enrico E Missirlis Fanis F Leimkühler Silke S

The Journal of experimental biology 20140415 Pt 12

In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are as ...[more]

PMID: 24737760

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Project description:Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain reliable results for the liberation of NADH during the initial-rate phase, the reaction was measured with a sensitive recording filter fluorimeter, and the complexes formed with the different dead-end and product inhibitors have been interpreted on the basis of a full dismutation reaction. The results are only consistent with a compulsory ordered reaction mechanism, with the formation of a dead-end binary enzyme-aldehyde complex. Under initial-velocity conditions, the rate of acetate release was calculated to be larger than 2.5 s-1, which is more than ten times that of NADH. The substrate specificity constant (kcat/Km or 1/phiB) with respect to the oxidation of substrates was propan-2-ol>ethanol>acetaldehyde>trimethylacetaldehyde.

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The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

Publications

The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

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