Four aromatic intradiol ring cleavage dioxygenases from Aspergillus niger.

Four aromatic intradiol ring cleavage dioxygenases from Aspergillus niger.

Appl Environ Microbiol. 2019 Sep 20;:

Authors: Semana P, Powlowski J

Abstract
Ring-cleavage dioxygenases catalyze the critical ring-opening step in the catabolism of aromatic compounds. The archetypal filamentous fungus, Aspergillus niger, previously has been reported to be able to utilize a range of monocyclic aromatic compounds as sole sources of carbon and energy. The genome of A. niger has been sequenced, and deduced amino acid sequences from a large number of gene models show varying levels of similarity to bacterial intradiol-cleavage dioxygenases, but no corresponding enzyme has been purified and characterized. Here, the cloning, heterologous expression, purification and biochemical characterization of four (encoded by NRRL3_02644, NRRL3_04787, NRRL3_05330 and NRRL3_01405) non-heme iron(III) containing intradiol dioxygenases from A. niger are reported. Purified enzymes were tested for their ability to cleave model catecholate substrates, and their apparent kinetic parameters were determined. Comparison of kcat/Km values show that NRRL3_02644 and NRRL3_05330 are specific for hydroxyquinol (1,2,4-trihydroxybenzene), and phylogenetic analysis shows that these two enzymes are related to bacterial hydroxyquinol 1,2-dioxygenases. A high activity catechol 1,2-dioxygenase (NRRL3_04787), which is phylogenetically related to other characterized and putative fungal catechol 1,2-dioxygenases, was also identified. The fourth enzyme (NRRL3_01405) appears to be a novel homodimeric Fe(III) containing protocatechuate 3,4-dioxygenase that is phylogenetically distantly related to heterodimeric bacterial protocatechuate 3,4-dioxygenases. These investigations provide experimental evidence for the molecular function of these proteins and open the way to further investigations of the physiological roles for these enzymes in fungal metabolism of aromatic compounds.Importance Aromatic ring-opening using molecular oxygen is one of the critical steps in the degradation of aromatic compounds by microorganisms. While enzymes catalyzing this step have been well -studied in bacteria, their counterparts from fungi are poorly characterized despite the abundance of genes annotated as ring-cleavage dioxygenases in fungal genomes. Aspergillus niger degrades a variety of aromatic compounds and its genome harbours 5 genes encoding putative intracellular intradiol dioxygenases. The ability to predict the substrate specificities of the encoded enzymes from sequence data is limited. We herein report the characterization of four purified intradiol ring-cleavage dioxygenases from A. niger, revealing two hydroxyquinol-specific dioxygenases, a catechol dioxygenase, and a unique homodimeric protocatechuate dioxygenase. Their characteristics, and phylogenetic relationships to predicted ring-cleavage dioxygenases from other fungal species, provide insights into their molecular functions in aromatic compound metabolism by this and other fungi.

PMID: 31540981 [PubMed – as supplied by publisher]

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