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Ring-cleaving dioxygenases with a cupin fold.

ABSTRACT: Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded ?-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an Fe(II) center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O(2), resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O(2) is supported by model chemistry.

SUBMITTER: Fetzner S

PROVIDER: S-EPMC3318818 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Ring-cleaving dioxygenases with a cupin fold.

Fetzner Susanne S

Applied and environmental microbiology 20120127 8

Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward the ...[more]

PMID: 22287012

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Project description:The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of Pseudomonas aeruginosa The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/β-hydrolase fold superfamily. The α/β-hydrolase fold core domain consists of a β-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the "nucleophilic elbow." In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of β-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.IMPORTANCE Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/β-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.

Project description:Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or "jelly roll" β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.

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

Ring-cleaving dioxygenases with a cupin fold.

Publications

Ring-cleaving dioxygenases with a cupin fold.

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