Extradiol Dioxygenase Research Articles

Regulate catalytic performance by engineering non-regular structure of extradiol dioxygenase: An entrance to bottom strategy

Extradiol dioxygenase Tcu3516 is a home-sourced enzyme demonstrating potent aromatic phenol degradation capacity. To add to the advantageous modifications inside active cavity, this work reported a novel strategy to engineer rarely concerned non-regular structures around the entrance towards the active site at the bottom of cavity. Three structures, Loop region 1 (Loop1: Met173-Arg185), Loop region 2 (Loop2: Ala201-Val212) and C-terminal (C-tail: His290-Lys306) were therefore identified through structural flexibility analysis. Highly rigid prolines within the structures were mutated into smaller alanine, glycine, or serine to improve structural flexibilities; while only P183S on Loop1 showed 3-fold activity enhancement vs the WT when subjected to cleavage of mono-cyclic catechol analogues. The analysis of Root Mean Square Fluctuation showed that P183S presents certain enhancement on Loop1 flexibility without dramatic changes of other domains. Furthermore, the synergetic effects from mutation P183S and cavity-based mutations V186L, V212N and D285A were evaluated by characterizing combinatorial mutants. Temperature dependence and thermostability of the combined mutants showed a more flexible catalytic domain without sacrificing structural integrity and stability. kcat value of P183S/V186L (SL) towards monocyclic catechols significantly surpasses any other combinatorial mutants around Tcu3516 active sites. Moreover, the synergetic effects on conformational plasticity were analyzed by molecular dynamic simulations to shed light into the interplay between structural changes and catalytic performance.

MEMO1 binds iron and modulates iron homeostasis in cancer cells.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

Discovery and characterization of l-DOPA 2,3-dioxygenase from Streptomyces hygroscopicus jingganensis

The largest natural reservoir of untapped carbon can be found in the cell-wall strengthening, plant woody-tissue polymer, lignin - a polymer of catechols or 1,2-dihydroxybenzene monomers. The catecholic carbon of lignin could be valorized into feedstocks and natural products by way of catabolic and biosynthetic transformations, including the oxygen-dependent cleavage reaction of extradiol dioxygenase (EDX) enzymes. The EDX l-DOPA 2,3-dioxygenase was first discovered as part of a biosynthetic gene cluster to the natural product antibiotic, lincomycin, and also contributes to the biosyntheses of anthramycin, sibiromycin, tomaymycin, porothramycin and hormaomycin. Using these l-DOPA 2,3-dioxygenases as a starting point, we searched sequence space in order to identify new sources of dioxygenase driven natural product diversity. A “vicinal-oxygen-chelate (VOC) family protein” from Streptomyces hygroscopicus jingganensis was identified using bioinformatic methods and biochemically investigated for dioxygenase activity against a suite of natural and synthetic catechols. Steady-state oxygen consumption assays were used to screen and identify substrates, and a steady-state kinetic model of oxygen consumption was developed to evaluate activity of the S. hygroscopicus jingganensis VOC-family-protein with respect to activity of l-DOPA 2,3-dioxygenases from Streptomyces lincolnensis and Streptomyces sclerotialus. Lastly, these data were integrated with steady-state kinetic methods to observe the formation of the EDX cleavage product with UV–visible spectroscopy. The genomic context and enzymatic activity of the S. hygroscopicus jingganensis VOC family protein are consistent with a l-DOPA 2,3-dioxygenase contained within a cryptic biosynthetic pathway.

Engineering non-conservative substrate recognition sites of extradiol dioxygenase: Computation guided design to diversify and accelerate degradation of aromatic compounds

Extradiol dioxygenases (EDOs) catalyzing meta-cleavage of catecholic compounds promise an effective way to detoxify aromatic pollutants. This work reported a novel scenario to engineer our recently identified Type I EDO from Tcu3516 for a broader substrate scope and enhanced activity, which was based on 2,3-dihydroxybiphenyl (2,3-DHB)-liganded molecular docking of Tcu3516 and multiple sequence alignment with other 22 Type I EDOs. 11 non-conservative residues of Tcu3516 within 6 Å distance to the 2,3-DHB ligand center were selected as potential hotspots and subjected to semi-rational design using 6 catecholic analogues as substrates; the mutants V186L and V212N returned with progressive evolution in substrate scope and catalytic activity. Both mutants were combined with D285A for construction of double mutants and final triple mutant V186L/V212N/D285A. Except for 2,3-DHB (the mutant V186L/D285A gave the best catalytic performance), the triple mutant prevailed all other 5 catecholic compounds for their degradation; affording the catalytic efficiency kcat/Km value increase by 10–30 folds, protein Tm (structural rigidity) increase by 15 °C and the half-life time enhancement by 10 times compared to the wild type Tcu3516. The molecular dynamic simulation suggested that a stabler core and a more flexible entrance are likely accounting for enhanced catalytic activity and stability of enzymes.

Efficient bio-remediation of multiple aromatic hydrocarbons using different types of thermotolerant, ring-cleaving dioxygenases derived from Aeribacillus pallidus HB-1

As toxic contaminants, aromatic compounds are widespread in most environmental matrices, and bioenzymatic catalysis plays a critical role in the degradation of xenobiotics. Here, a thermophillic aromatic hydrocarbon degrader Aeribacillus pallidus HB-1 was found. Bioinformatic analysis of the HB-1 genome revealed two ring-cleaving extradiol dioxygenases (EDOs), among which, EDO-0418 was assigned to a new subfamily of type I.1 EDOs and exhibited a broad substrate specificity, particularly towards biarylic substrate. Both EDOs exhibited optimal activities at elevated temperatures (55 and 65 °C, respectively) and showed remarkable thermostability, pH stability, metal ion resistance and tolerance to chemical reagents. Most importantly, simulated wastewater bioreactor experiments demonstrated efficient and uniform degradation performance of mixed aromatic substrates under harsh environments by the two enzymes combined for potential industrial applications. The unveiling of two thermostable dioxygenases with broad substrate specificities and stress tolerance provides a novel approach for highly efficient environmental bioremediation using composite enzyme systems.

Genomic insights on gene clusters and pathways for the biodegradation of plastic compounds: Unravelling the metabolic versatility in a Dietzia kunjamensis IITR165

The Dietzia kunjamensis IITR165 bacterium, capable of degrading dibutyl phthalate (DBP), terephthalate (TPA), and polyethylene terephthalate (PET), was studied to uncover its metabolic pathways. Whole-genome analysis revealed a circular chromosome of 3,477,711 bp and a plasmid of 58,850 bp with 70.6 % GC content. Among 3,311 functional genes, phthalate dioxygenase/decarboxylase (padAa1, padAb1, phtB, phtC), alkane monooxygenase (alkB), di-and mono-alkyl phthalate hydrolase, and extra-diol dioxygenase were identified. Gene clusters for terephthalate (tphA1A2A3 and tphB), benzoic acid (benABCD), and catechol (catABCD) were also found. Strain IITR165 metabolized of 1000 mg/L of TPA in 96 h with a half-life of 15.36 h−1, producing phthalic acid (PA), benzoic acid (BA), and catechol as metabolites based on Q-TOF LC/MS-MS analysis. Scanning electron micrograph reveals the extensive biofilm and surface modification of PET sheet after bacterial treatment. A novel PET-hydrolase (PET165) protein, sharing 45.70 % amino acid homology with reported PETases, was discovered, with docking studies showing a conserved catalytic triad (Serine-128, Aspartate-261, and Histidine-287) interacting with the PET ligand. The presence of this novel PET hydrolase and the tpa gene cluster, along with genes involved in nylon, and polystyrene metabolism, indicates versatility of the bacterium useful in treatment of a mixed plastic contaminated ecological niches.

Structure and biochemical characterization of an extradiol 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Acinetobacter baumannii

3,4-Dihydroxyphenylacetate (DHPA) 2,3-dioxygenase (EC 1.13.11.15) from Acinetobacter baumannii (AbDHPAO) is an enzyme that catalyzes the 2,3-extradiol ring-cleavage of DHPA in the p-hydroxyphenylacetate (HPA) degradation pathway. While the biochemical reactions of various DHPAOs have been reported, only structures of DHPAO from Brevibacterium fuscum and their homologs are available. Here, we report the X-ray structure and biochemical characterization of an Fe2+-specific AbDHPAO that shares 12% sequence identity to the enzyme from B. fuscum. The 1.8 Å X-ray structure of apo-AbDHPAO was determined with four subunits per asymmetric unit, consistent with a homotetrameric structure. Interestingly, the αβ-sandwiched fold of the AbDHPAO subunit is different from the dual β-barrel-like motif of the well-characterized B. fuscum DHPAO structures; instead, it is similar to the structures of non-DHPA extradiol dioxygenases from Comamonas sp. and Sphingomonas paucimobilis. Similarly, these extradiol dioxygenases share the same chemistry owing to a conserved 2-His-1-carboxylate catalytic motif. Structure analysis and molecular docking suggested that the Fe2+ cofactor and substrate binding sites consist of the conserved residues His12, His57, and Glu238 forming a 2-His-1-carboxylate motif ligating to Fe2+ and DHPA bound with Fe2+ in an octahedral coordination. In addition to DHPA, AbDHPAO can also use other 3,4-dihydroxyphenylacetate derivatives with different aliphatic carboxylic acid substituents as substrates, albeit with low reactivity. Altogether, this report provides a better understanding of the structure and biochemical properties of AbDHPAO and its homologs, which is advancing further modification of DHPAO in future applications.

Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Comparison of betalain compounds in two Beta vulgaris var. cicla and BvCYP76AD27 function identification in betalain biosynthesis

Beta vulgaris var. cicla is an edible, ornamental and horticultural plant. However, the difference of components and contents of betalain in beets with different leaf color are not well understood. Here, the stress resistance and metabolites of two B. vulgaris var. cicla cultivars were determined. The differences in stress resistance between red leaf-colored chard (RC) and yellow leaf-colored chard (YC) were positively related to betacyanins (BC) and betaxathins (BX) content in the leaves. Furthermore, a total of 3615 distinct metabolites were identified by UPLC-QTOF-MS in two cultivars, including 70 alkaloids and their derivatives, 249 flavonoids, and 264 terpenoids. There were 17 metabolites attributed to betalain biosynthesis pathway, seven of nine BC were up-regulated, and eight BX showed no significant difference in RC compared with YC. The contents of celosianin II and betanin were the highest BC in RC, at approximately 84.38 and 19.97 times that of YC, respectively. The content of portulacaxanthin II was the highest BX in two beets. Additionally, the BvCYP450 genes were identified based on genome, and the members that might be involved in betalain biosynthesis were screened. BvCYP76AD27, a member of the BvCYP76AD subfamily, had a higher expression level in RC than YC under freezing, drought and shading stress. In yeast Saccharomyces cerevisiae, BvCYP76AD5 and BvCYP76AD27 only hydroxylated tyrosine to L-DOPA, which was transformed into portulacaxanthin II by 4,5-DOPA extradiol dioxygenase. The results contribute to illustrating the molecular mechanism of betalain biosynthesis and provide useful information for further investigation of beet chemistry and sufficient utilization of this species.

Correction to: Biochemical and genetic characterization comparison of four extradiol dioxygenases in Rhizorhabdus wittichii RW1

Correction to: Biochemical and genetic characterization comparison of four extradiol dioxygenases in Rhizorhabdus wittichii RW1

Enzyme adaptation to habitat thermal legacy shapes the thermal plasticity of marine microbiomes

Microbial communities respond to temperature with physiological adaptation and compositional turnover. Whether thermal selection of enzymes explains marine microbiome plasticity in response to temperature remains unresolved. By quantifying the thermal behaviour of seven functionally-independent enzyme classes (esterase, extradiol dioxygenase, phosphatase, beta-galactosidase, nuclease, transaminase, and aldo-keto reductase) in native proteomes of marine sediment microbiomes from the Irish Sea to the southern Red Sea, we record a significant effect of the mean annual temperature (MAT) on enzyme response in all cases. Activity and stability profiles of 228 esterases and 5 extradiol dioxygenases from sediment and seawater across 70 locations worldwide validate this thermal pattern. Modelling the esterase phase transition temperature as a measure of structural flexibility confirms the observed relationship with MAT. Furthermore, when considering temperature variability in sites with non-significantly different MATs, the broadest range of enzyme thermal behaviour and the highest growth plasticity of the enriched heterotrophic bacteria occur in samples with the widest annual thermal variability. These results indicate that temperature-driven enzyme selection shapes microbiome thermal plasticity and that thermal variability finely tunes such processes and should be considered alongside MAT in forecasting microbial community thermal response.

Combined Omics Approach Reveals Key Differences between Aerobic and Microaerobic Xylene-Degrading Enrichment Bacterial Communities: Rhodoferax─A Hitherto Unknown Player Emerges from the Microbial Dark Matter.

Among monoaromatic hydrocarbons, xylenes, especially the ortho and para isomers, are the least biodegradable compounds in oxygen-limited subsurface environments. Although much knowledge has been gained regarding the anaerobic degradation of xylene isomers in the past 2 decades, the diversity of those bacteria which are able to degrade them under microaerobic conditions is still unknown. To overcome this limitation, aerobic and microaerobic xylene-degrading enrichment cultures were established using groundwater taken from a xylene-contaminated site, and the associated bacterial communities were investigated using a polyphasic approach. Our results show that the xylene-degrading bacterial communities were distinctly different between aerobic and microaerobic enrichment conditions. Although members of the genus Pseudomonas were the most dominant in both types of enrichments, the Rhodoferax and Azovibrio lineages were only abundant under microaerobic conditions, while Sphingobium entirely replaced them under aerobic conditions. Analysis of a metagenome-assembled genome of a Rhodoferax-related bacterium revealed aromatic hydrocarbon-degrading ability by identifying two catechol 2,3-dioxygenases in the genome. Moreover, phylogenetic analysis indicated that both enzymes belonged to a newly defined subfamily of type I.2 extradiol dioxygenases (EDOs). Aerobic and microaerobic xylene-degradation experiments were conducted on strains Sphingobium sp. AS12 and Pseudomonas sp. MAP12, isolated from the aerobic and microaerobic enrichments, respectively. The obtained results, together with the whole-genome sequence data of the strains, confirmed the observation that members of the genus Sphingobium are excellent aromatic hydrocarbon degraders but effective only under clear aerobic conditions. Overall, it was concluded that the observed differences between the bacterial communities of aerobic and microaerobic xylene-degrading enrichments were driven primarily by (i) the method of aromatic ring activation (monooxygenation vs dioxygenation), (ii) the type of EDO enzymes, and (iii) the ability of degraders to respire utilizing nitrate.

Bioconversion of 4-hydroxyestradiol by extradiol ring-cleavage dioxygenases from Novosphingobium sp. PP1Y

Livestock breeding activities and pharmaceutical wastes lead to considerable accumulation of steroid hormones and estrogens in wastewaters. Here estrogens act as pro-cancerogenic agents and endocrine disruptors interfering with the sexual development of aquatic animals and having toxic effects in humans. Environmental bacteria play a vital role in estrogens degradation. Their wide reservoir of enzymes, such as ring cleavage dioxygenases (RCDs), can degrade the steroid nucleus, catalyzing the meta-cleavage of A, B or D steroid rings. In this work, 4 extra-diol ring cleavage dioxygenases (ERCDs), PP28735, PP26077, PP00124 and PP00193, were isolated from the marine sphingomonad Novosphingobium sp. PP1Y and characterized. Enzymes kinetic parameters were determined on different synthetic catecholic substrates. Then, the bioconversion of catechol estrogens was evaluated. PP00124 showed to be an efficient catalyst for the degradation of 4-hydroxyestradiol (4-OHE2), a carcinogenic hydroxylated derivate of E2. 4-OHE2 complete cleavage was obtained using PP00124 both in soluble form and in whole recombinant E. coli cells. LC–MS/MS analyses confirmed the generation of a semialdehyde product, through A-ring meta cleavage. To the best of our knowledge, PP00124 is the first characterized enzyme able to directly degrade 4-OHE2 via meta cleavage. Moreover, the complete 4-OHE2 biodegradation using recombinant whole cells highlighted advantages for bioremediation purposes.

Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol-Degrading Haloalkaliphilic Bacterial Isolates

Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.

Biochemical and genetic characterization comparison of four extradiol dioxygenases in Rhizorhabdus wittichii RW1.

Rhizorhabdus (previously Sphingomonas) wittichii RW1 uses a diverse array of aromatic organic compounds as energy and carbon sources, including some extremely recalcitrant compounds such as dibenzo-p-dioxin and dibenzofuran. Extradiol dioxygenases play a key role in the metabolism of dibenzofuran (DBF), dibenzo-p-dioxin (DBD), PCBs, and various other aromatic compounds. In this study, a detailed kinetic analysis of four extradiol dioxygenases identified in R. wittichii RW1 (DbfB, Edo2, Edo3, and Edo4) showed all of them to be typical 2,3dihydroxybiphenyl (DHB) dioxygenases with DHB as preferred substrate (kcat/Km values of 0.13-188 (µM -1s-1)) and only slightly lower activity against trihydroxybiphenyl (THB) whereas monocyclic substrates were, to different extents, poor substrates due to high km values. All extradiol dioxygenases analyzed were subject to mechanism-based inactivation by 2,2`,3-trihydroxybiphenylether (THBE) the intermediate of DBD degradation. However, Edo4 was superior as reflected by the relatively high partition ratio and the comparably low efficiency of inactivation. Significant differences were observed with respect to their inactivation by 3-chlorocatechol. The absence of any significant mechanism-based inactivation makes Edo3 a perfect candidate for being recruited for chlorobiphenyl degradation where inactivation of extradiol dioxygenases by this intermediate creates significant metabolic problems. KEY POINTS: • Characterization of additional extradiol dioxygenases encoded by RW1 • Identification of differences in 2,2`,3-trihydroxybiphenylether transformation • Identification of differences in inhibition by 3-chlorocatechol.

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases.

Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

Derivatives of 3,4‐dihydroxyhydrocinnamic acid at the 6‐position as mechanistic probes of L‐DOPA dioxygenase

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials as well as degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these powerful enzymes. To this end, we report the syntheses, redox potentials and pKa s of 3,4‐dihydroxyhydrocinnamic acid (DHHCA) derivatives substituted at the 6‐position and their characterization as substrates of L‐DOPA dioxygenase from Streptomyces lincolnensis. The cleavage of diverse catecholic substrates is an important element of bioremediation. Extradiol dioxygenase cleavage of DHHCA derivatives also promises to yield insight into mechanism and provide synthons for various applications.

Formation of carboxylated and decarboxylated betalains in ripening grains of Chenopodium quinoa by a dual dioxygenase.

Chenopodium quinoa (quinoa) is a pseudo-cereal that forms part of the cultural heritage of Andean countries, and its grains have high nutritional value and potential health benefits. Betalains are nitrogenous water-soluble pigments and bioactive molecules that contribute to these health-promoting properties. Betalains are restricted to plants of the order Caryophyllales, to which quinoa belongs. A new family of betalains has been discovered in the form of unconventional decarboxylated pigments. Here, we show that these pigments accumulate in ripening quinoa grains of fluorescent nature, and are putatively based on a dopamine-cleaving activity. This study describes for the first time the purification and molecular and functional characterization of a 4,5-dopamine extradiol dioxygenase enzyme from plants. It is a monomeric protein with a molecular mass of 34.5 kDa characterized by chromatography, electrophoresis, and time-of-flight mass spectrometry. We demonstrate that this key enzyme has a dual function in a square-shaped biosynthetic pathway towards the formation of both carboxylated and decarboxylated pigments. Enzyme kinetic properties are characterized for the production of 6-decarboxy-betalamic acid and 3,4-dihydroxy-l-phenylalanine-derived betalamic acid, the two structural units of plant pigment in nature. The profile of multiple betalains present in quinoa grains has been reproduced in one-pot bioreactors containing the novel enzyme and two competing substrates.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M−1 s−1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates’ binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate’s alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

Bioconversion of lignin-derived aromatics into the building block pyridine 2,4-dicarboxylic acid by engineering recombinant Pseudomonas putida strains

2,4 pyridine dicarboxylic acid (2,4 PDCA) is an analogue of terephthalate, and hence a target chemical in the field of bio-based plastics. Here, Pseudomonas putida KT2440 strains were engineered to efficiently drive the metabolism of lignin-derived monoaromatics towards 2,4 PDCA in a resting cells-based bioprocess that alleviates growth-coupled limitations and allows biocatalysts recycling. Native β-ketoadipate pathway was blocked by replacing protocatechuate 3,4-dioxygenase by the exogenous LigAB extradiol dioxygenase. Overexpression of pcaK encoding a transporter increased 8-fold 2,4 PDCA productivity from protocatechuate, reaching the highest value reported so far (0.58 g L-1h−1). Overexpression of the 4-hydroxybenzoate monooxygenase (pobA) speed up drastically the production of 2,4 PDCA from 4-hydroxybenzoate (0.056 g L-1h−1) or p-coumarate (0.012 g L-1h−1) achieving values 15-fold higher than those reported with Rhodococcus jostii biocatalysts. 2,4 PDCA was also bioproduced by using soda lignin as feedstock, paving the way for future polymeric lignin valorization approaches.