Four Aromatic Intradiol Ring Cleavage Dioxygenases from Aspergillus niger

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 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/K_m 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.

Keywords: Aspergillus niger; aromatic compounds; dioxygenases.

FIG 1

Multiple-sequence alignment of amino acid sequences of proteins in this study with characterized members of the catechol 1,2-dioxygenase superfamily. The sequences are denoted by the assigned IDs as described in this study. Sequences used, with the corresponding UniProt IDs, are the following: P_putida_HXQ1_2D, hydroxyquinol 1,2-dioxygenase from Pseudomonas putida, ID C6FI44; R_opacus_HXQ1,2-D, hydroxyquinol 1,2-dioxygenase from Rhodococcus opacus, ID Q6F4M7; C_albicans_Cat1,2-D, catechol 1,2-dioxygenase from Candida albicans, ID P86029; A_sp_ADP1_Cat1,2-D, catechol 1,2-dioxygenase from Pseudomonas sp. strain EST1001, ID P31019; and Lwoffi_Cat1,2-D, catechol 1,2-dioxygenase from Acinetobacter lwoffii, ID O33950. The sequence alignment was made with ClustalOmega, and the figure was generated by using Esprit (29, 58).

FIG 2

Cartoon representation of the homology-based model of the protein encoded by NRRL3_04787. The model was generated by SWISS-Model based on the homology to dimeric catechol 1,2-dioxygenase from Pseudomonas arvilla (PDB entry 2AZQ) (59). The inset shows a zoom-in of the predicted active-site Fe(III) and the four structurally conserved residues. The figure was prepared using PyMOL (46).

FIG 3

Coomassie blue-stained 12% SDS-PAGE gel showing the expression and purification achieved for the enzymes used in this study. Lane 1 is the low-molecular-weight marker, and lanes 3, 5, 7, and 9 are 2 μg of purified proteins encoded by NRRL3_01405, NRRL3_05330, NRRL3_02644, and NRRL3_04787, respectively. For comparison, the adjacent lanes were loaded with soluble crude extracts (20 μg) from E. coli BL21(DE3) expressing the corresponding proteins.

FIG 4

UV-visible absorption spectrum of purified enzyme encoded by NRRL3_04787 (15.5 mg/ml in 50 mM Tris-HCl, pH 7.6, at 25°C).

FIG 5

UV-visible absorption spectral changes during intradiol cleavage of catechol by the NRRL3_02644-encoded enzyme (a) and extradiol cleavage of catechol by the NRRL3_02644-encoded enzyme (b). Absorbance spectra were recorded every 60 s after the addition of 10 μg of purified enzyme for catechol and 20 μg for 3-methylcatechol at room temperature. The inset arrows show the direction of increase in absorbance. The dashed-line spectra were recorded with substrates only (for 3 min) before adding the enzyme.

FIG 6

Saturation kinetics of the dioxygenase encoded by NRRL3_04787 on catechol (a) and 4-methylcatechol (b). The graphs show initial rates plotted as a function of various substrate concentrations (squares) and the fit to the Michaelis-Menten equation (continuous line) using OriginLab. The best-fit values for K_m and V_max were 2.4 μM and 0.0051 μmol/min for catechol as a substrate, respectively. For 4-methycatechol the K_m was 45.8 μM and V_max was 0.0025 μmol/min (enzyme concentration, 9.7 nM). The error bars are standard deviations for three replicate values. Activity assays were carried out as described in Materials and Methods with various substrate concentrations.

FIG 7

UV region spectra recorded during the enzymatic conversion of 3,4-dihydroxybenzoic acid to 3-carboxy-cis,cis-muconate by the enzyme encoded by NRRL3_01405. The spectra were recorded every 60 s after adding 10 μg of purified enzyme to 100 μM 3,4-dihydroxybenzoic acid in a final volume of 50 mM Tris-HCl, pH 7.6, buffer at room temperature. The inset arrows indicate the direction of change in absorbance.

FIG 8

(a) Sedimentation velocity analytical ultracentrifugation of purified NRRL3_01405 dioxygenase. A snapshot of the DCDT+ analysis results of the AUC data are shown, with the upper panel showing the g(s*) plot and the lower panel showing the residuals from the sedimentation boundary analysis and fitted parameters (57). (b) SDS-PAGE electrophoretic analysis of the EDC cross-linking reactions of purified proteins (10 μg) encoded by NRRL3_01405 (lane 1), NRRL3_02644 (lane 2), NRRL3_05330 (lane 3), and NRRL3_04787 (lane 4).

FIG 9

Neighbor-joining phylogenetic tree of the A. niger intradiol ring cleavage dioxygenases (encoded by NRRL3_01405, NRRL3_02644, NRRL3_04787, and NRRL3_05330) and their homologs from bacterial and fungal species. The tree was constructed using MEGA6, and sequence alignments were performed using ClustalW incorporated in MEGA6 (30). Sequences are named by their corresponding NBCI GenBank gi numbers followed by the species names. 3,4-PCD, protocatechuate 3,4-dioxygenases; Cat 1,2-D, catechol 1,2-dioxgenases; HXQ 1,2-D, hydroxyquinol 1,2-dioxygenases.