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Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase.

ABSTRACT: Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe(3+) to activate O2 and catecholic substrates for reaction. The inability of Fe(3+) to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe(3+) species, and the anhydride-Fe(3+) intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe(2+)-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe(2+) intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

SUBMITTER: Knoot CJ

PROVIDER: S-EPMC4299229 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase.

Knoot Cory J CJ Purpero Vincent M VM Lipscomb John D JD

Proceedings of the National Academy of Sciences of the United States of America 20141229 2

Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe(3+) to activate O2 and catecholic substrates for reaction. The inability of Fe(3+) to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies ...[more]

PMID: 25548185

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Project description: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 various levels of similarity to bacterial intradiol ring cleavage dioxygenases, but no corresponding enzyme has been purified and characterized. Here, the cloning, heterologous expression, purification, and biochemical characterization of four nonheme iron(III)-containing intradiol dioxygenases (NRRL3_02644, NRRL3_04787, NRRL3_05330, and NRRL3_01405) from A. niger are reported. Purified enzymes were tested for their ability to cleave model catecholate substrates, and their apparent kinetic parameters were determined. Comparisons of k cat /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 harbors 5 genes encoding putative intracellular intradiol dioxygenases. The ability to predict the substrate specificities of the encoded enzymes from sequence data are limited. Here, we 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, as well as their phylogenetic relationships to predicted ring cleavage dioxygenases from other fungal species, provide insights into their molecular functions in aromatic compound metabolism by this fungus and other fungi.

Dataset Information

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase.

Publications

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase.

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