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Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t -anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t -anethole oxygenase from Stenotrophomonas maltophilia (TAO Sm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAO Sm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t -anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAO Sm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAO Sm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry. • Identification of a new class of heme-containing enzymes. • Cofactor-independent biocatalytic C C cleavage. • Crystal structure of a trans-anethole oxygenase. • Novel active site architecture of a heme-containing enzyme. [ABSTRACT FROM AUTHOR]

By demethylating or clipping histones, Jumonji C domain-containing (JMJD) proteins induce chromatin changes that lie beneath the epigenetic control of gene expression. Through a wide repertoire of catalytic activities (hydroxylation, demethylation, proteolytic cleavage) and non-catalytic activities affecting non-histone proteins, JMJD proteins shape various cellular processes beyond epigenetics. Several oncometabolites, oxygen tension, reactive oxygen species (ROS), and heavy metals all affect JMJD catalytic activity, allowing JMJD proteins to sense metabolic changes. Pathogenic JMJD mutations or dysregulated JMJD activity underlie various diseases, including cancer and neurodevelopmental defects, and may also account for sex-dependent differences in disease prevalence and manifestation. Inhibiting or activating specific JMJD proteins may be efficacious in clinical therapy. Jumonji C domain-containing (JMJD) proteins are found in bacteria, fungi, animals, and plants. They belong to the 2-oxoglutarate-dependent oxygenase superfamily and are endowed with various enzymatic activities, including demethylation of histones and hydroxylation of non-histone proteins. Many JMJD proteins are involved in the epigenetic control of gene expression, yet they also modulate a myriad other cellular processes. In this review we focus on the 33 human JMJD proteins and their established and controversial catalytic properties, survey their epigenetic and non-epigenetic functions, emphasize their contribution to sex-specific disease differences, and highlight how they sense metabolic changes. All this underlines not only their key roles in development and homeostasis, but also that JMJD proteins are destined to become drug targets in multiple diseases. [ABSTRACT FROM AUTHOR]

Indole and skatole (3-methylindole, C9H9N) are common nitrogen-containing heterocyclic pollutants found in waste, wastewater treatment plants, and public restrooms and are the most notorious compounds in animal feces. Biodegradation was considered a feasible method for the removal of indole and skatole, but a comprehensive understanding of the metabolic pathways under both aerobic and anaerobic conditions was lacking, and the functional genes responsible for skatole biodegradation remained a mystery. Through metagenomic and gene cluster functional analysis, Acinetobacter piscicola p38 (NCBI: CP167896), genes 1650 (styrene monooxygenase: ACDW34_08180), and 1687 (styrene monooxygenase: ACDW34_08350) were identified as having the potential to degrade indole and skatole. The heterologous expression results demonstrate that the genes 1650 and 1651 (flavin reductase: ACDW34_08185), when combined, are capable of degrading indole, while the genes 1687 and 1688 (flavin reductase: ACDW34_08355), in combination, can degrade indole as well as skatole. These reactions necessitate the involvement of flavin reductase and NAD(P)H to catalyze the oxygenation process. This work aimed to provide new experimental evidence for the biodegradation of indole and skatole. This study offered new insights into our understanding of skatole degradation. The Acinetobacter_piscicola p38 strain provided an effective bacterial resource for the bioremediation of fecal indole and skatole. [ABSTRACT FROM AUTHOR]

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. Key points: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified. • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin. [ABSTRACT FROM AUTHOR]

Indole and skatole (3-methylindole, C9H9N) are common nitrogen-containing heterocyclic pollutants found in waste, wastewater treatment plants, and public restrooms and are the most notorious compounds in animal feces. Biodegradation was considered a feasible method for the removal of indole and skatole, but a comprehensive understanding of the metabolic pathways under both aerobic and anaerobic conditions was lacking, and the functional genes responsible for skatole biodegradation remained a mystery. Through metagenomic and gene cluster functional analysis, Acinetobacter piscicola p38 (NCBI: CP167896), genes 1650 (styrene monooxygenase: ACDW34_08180), and 1687 (styrene monooxygenase: ACDW34_08350) were identified as having the potential to degrade indole and skatole. The heterologous expression results demonstrate that the genes 1650 and 1651 (flavin reductase: ACDW34_08185), when combined, are capable of degrading indole, while the genes 1687 and 1688 (flavin reductase: ACDW34_08355), in combination, can degrade indole as well as skatole. These reactions necessitate the involvement of flavin reductase and NAD(P)H to catalyze the oxygenation process. This work aimed to provide new experimental evidence for the biodegradation of indole and skatole. This study offered new insights into our understanding of skatole degradation. The Acinetobacter_piscicola p38 strain provided an effective bacterial resource for the bioremediation of fecal indole and skatole.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron (II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization's list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues' contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst. [ABSTRACT FROM AUTHOR]

Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila, NcAA13 and MtAA13, respectively, on three different starch substrates are reported here. Using high-performance anion-exchange chromatography coupled with pulsed amperometry detection, we observed that both enzymes have significantly higher oxidative activity on amylose than on amylopectin and cornstarch. Analysis of the product distribution revealed that NcAA13 and MtAA13 more frequently oxidize glycosidic linkages separated by multiples of a helical turn consisting of six glucose units on the same amylose helix. Docking studies identified important residues that are involved in amylose binding and suggest that the shallow groove that spans the active-site surface of AA13 PMOs favors the binding of helical amylose substrates over nonhelical substrates. Truncations of NcAA13 that removed its native carbohydrate-binding module resulted in diminished binding to amylose, but truncated NcAA13 still favored amylose oxidation over other starch substrates. These findings establish that AA13 PMOs preferentially bind and oxidize the helical starch substrate amylose. Moreover, the product distributions of these two enzymes suggest a unique interaction with starch substrates.

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron (II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (−)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.

CADD (chlamydia protein associating with death domains) is a p‐aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope‐labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry‐based proteomic analyses of CADD‐derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor. [ABSTRACT FROM AUTHOR]

Inositol depletion has been associated with diabetes and related complications. Increased inositol catabolism, via myo-inositol oxygenase (MIOX), has been implicated in decreased renal function. This study demonstrates that the fruit fly Drosophila melanogaster catabolizes myo-inositol via MIOX. The levels of mRNA encoding MIOX and MIOX specific activity are increased when fruit flies are grown on a diet with inositol as the sole sugar. Inositol as the sole dietary sugar can support D. melanogaster survival, indicating that there is sufficient catabolism for basic energy requirements, allowing for adaptation to various environments. The elimination of MIOX activity, via a piggyBac WH-element inserted into the MIOX gene, results in developmental defects including pupal lethality and pharate flies without proboscises. In contrast, RNAi strains with reduced levels of mRNA encoding MIOX and reduced MIOX specific activity develop to become phenotypically wild-type-appearing adult flies. myo-Inositol levels in larval tissues are highest in the strain with this most extreme loss of myo-inositol catabolism. Larval tissues from the RNAi strains have inositol levels higher than wild-type larval tissues but lower levels than the piggyBac WH-element insertion strain. myo-Inositol supplementation of the diet further increases the myo-inositol levels in the larval tissues of all the strains, without any noticeable effects on development. Obesity and blood (hemolymph) glucose, two hallmarks of diabetes, were reduced in the RNAi strains and further reduced in the piggyBac WH-element insertion strain. Collectively, these data suggest that moderately increased myo-inositol levels do not cause developmental defects and directly correspond to reduced larval obesity and blood (hemolymph) glucose. [ABSTRACT FROM AUTHOR]

Rubisco catalysis is complex and includes an activation step through the formation of a carbamate at the conserved active site lysine residue and the formation of a highly reactive enediol that is the key to its catalytic reaction. The formation of this enediol is both the basis of its success and its Achilles' heel, creating imperfections to its catalytic efficiency. While Rubisco originally evolved in an atmosphere of high CO2, the earth's multiple oxidation events provided challenges to Rubisco through the fixation of O2 that competes with CO2 at the active site. Numerous catalytic screens across the Rubisco superfamily have identified significant variation in catalytic properties that have been linked to large and small subunit sequences. Despite this, we still have a rudimentary understanding of Rubisco's catalytic mechanism and how the evolution of kinetic properties has occurred. This review identifies the lysine base that functions both as an activator and a proton abstractor to create the enediol as a key to understanding how Rubisco may optimize its kinetic properties. The ways in which Rubisco and its partners have overcome catalytic and activation imperfections and thrived in a world of high O2, low CO2, and variable climatic regimes is remarkable. [ABSTRACT FROM AUTHOR]

Oxygenases, which catalyze the reductive activation of O2 and incorporation of oxygen atoms into substrates, are widely distributed in aerobes. They function by switching the redox states of essential cofactors that include flavin, heme iron, Rieske non-heme iron, and Fe(II)/α-ketoglutarate. This review summarizes the catalytic features of flavin-dependent monooxygenases, heme iron-dependent cytochrome P450 monooxygenases, Rieske non-heme iron-dependent oxygenases, Fe(II)/α-ketoglutarate-dependent dioxygenases, and ring-cleavage dioxygenases, which are commonly involved in pesticide degradation. Heteroatom release (hydroxylation-coupled hetero group release), aromatic/heterocyclic ring hydroxylation to form ring-cleavage substrates, and ring cleavage are the main chemical fates of pesticides catalyzed by these oxygenases. The diversity of oxygenases, specificities for electron transport components, and potential applications of oxygenases are also discussed. This article summarizes our current understanding of the catalytic mechanisms of oxygenases and a framework for distinguishing the roles of oxygenases in pesticide degradation. [ABSTRACT FROM AUTHOR]

Oxygenases, which catalyze the reductive activation of O2 and incorporation of oxygen atoms into substrates, are widely distributed in aerobes. They function by switching the redox states of essential cofactors that include flavin, heme iron, Rieske non-heme iron, and Fe(II)/α-ketoglutarate. This review summarizes the catalytic features of flavin-dependent monooxygenases, heme iron–dependent cytochrome P450 monooxygenases, Rieske non-heme iron–dependent oxygenases, Fe(II)/α-ketoglutarate-dependent dioxygenases, and ring-cleavage dioxygenases, which are commonly involved in pesticide degradation. Heteroatom release (hydroxylation-coupled hetero group release), aromatic/heterocyclic ring hydroxylation to form ring-cleavage substrates, and ring cleavage are the main chemical fates of pesticides catalyzed by these oxygenases. The diversity of oxygenases, specificities for electron transport components, and potential applications of oxygenases are also discussed. This article summarizes our current understanding of the catalytic mechanisms of oxygenases and a framework for distinguishing the roles of oxygenases in pesticide degradation. [ABSTRACT FROM AUTHOR]

The widespread use of plastics has led to their increasing presence in the environment and subsequent pollution. Some microorganisms degrade plastics in natural ecosystems and the associated metabolic pathways can be studied to understand the degradation mechanisms. Polystyrene (PS) is one of the more recalcitrant plastic polymers that is degraded by only a few bacteria. Exiguobacterium is a genus of Gram-positive poly-extremophilic bacteria known to degrade PS, thus being of biotechnological interest, but its biochemical mechanisms of degradation have not yet been elucidated. Based solely on genome annotation, we initially proposed PS degradation by Exiguobacterium sp. RIT 594 via depolymerization and epoxidation catalyzed by a ring epoxidase. However, Fourier transform infrared (FTIR) spectroscopy analysis revealed an increase of carboxyl and hydroxyl groups with biodegradation, as well as of unconjugated C-C double bonds, both consistent with dearomatization of the styrene ring. This excludes any aerobic pathways involving side chain epoxidation and/or hydroxylation. Subsequent experiments confirmed that molecular oxygen is critical to PS degradation by RIT 594 because degradation ceased under oxygen-deprived conditions. Our studies suggest that styrene breakdown by this bacterium occurs via the sequential action of two enzymes encoded in the genome: an orphan aromatic ring-cleaving dioxygenase and a hydrolase. [ABSTRACT FROM AUTHOR]

Halogenated organic compounds are naturally occurring in subsurface environments; however, accumulation of the degradative intermediate cis-1,2-dichloroethene (cDCE) at soil and groundwater sites contaminated with xenobiotic chlorinated ethenes is a global environmental and public health issue. Identifying microorganisms capable of cDCE degradation in these environments is of interest because of their potential application to bioremediation techniques. In this study, we sequenced, assembled, and analyzed the complete genome of Acinetobacter pittii CEP14, a strain isolated from chloroethene-contaminated groundwater, that has demonstrated the ability for aerobic cometabolic degradation of cDCE in the presence of n-hexane, phenol, and toluene. The A. pittii CEP14 genome consists of a 3.93 Mbp-long chromosome (GenBank accession no. CP084921) with a GC content of 38.9% and three plasmids (GenBank accession no. CP084922, CP084923, and CP084924). Gene function was assigned to 83.4% of the 3,930 coding DNA sequences. Functional annotation of the genome revealed that the CEP14 strain possessed all genetic elements to mediate the degradation of a range of aliphatic and aromatic compounds, including n-hexane and phenol. In addition, it harbors gene clusters involved in cytosol detoxification and oxidative stress resistance, which could play a role in the mitigation of toxic chemical intermediates that can arise during the degradation of cDCE. Gene clusters for heavy metal and antibiotic resistance were also identified in the genome of CEP14. These results suggest that CEP14 may be a versatile degrader of xenobiotic compounds and well-adapted to polluted environments, where a combination of heavy metal and organic compound pollution is often found. [ABSTRACT FROM AUTHOR]

Caryoynencin is a toxic and antifungal fatty acid derivative produced by a number of plant‐pathogenic and insect‐protective bacteria (Trinickia caryophylli and Burkholderia spp.). In addition to the reactive tetrayne unit, the presence of an allylic alcohol moiety is critical for antimicrobial activities. By a combination of mutational analyses, heterologous expression and in vitro reconstitution experiments we show that the cytochrome P450 monooxygenase CayG catalyzes the complex transformation of a saturated carbon backbone into an allylic alcohol. Unexpectedly, CayG employs a ferritin‐like protein (CayK) or a rubredoxin (CayL) component for electron transport. A desaturation‐hydroxylation sequence was deduced from a time‐course study and in vitro biotransformations with pathway intermediates, substrate analogues, protegencin congeners from Pseudomonas protegens Pf‐5, and synthetic derivatives. This unusual multifunctional oxygenase may inspire future biocatalytic applications. [ABSTRACT FROM AUTHOR]

Several bacteria possess components of catabolic pathways for the synthetic polyester poly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages of the polymer to its monomers, ethylene glycol and terephthalate (TPA), which are further converted into common metabolites. These pathways are crucial for genetically engineering microbes for PET upcycling, prompting interest in their fundamental biochemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cognate reductase make up a complex multimetalloenzyme system that dihydroxylates TPA, activating it for enzymatic decarboxylation to yield protocatechuic acid (PCA). Here, we report structural, biochemical, and bioinformatic analyses of TPADO. Together, these data illustrate the remarkable adaptation of TPADO to the TPA dianion as its preferred substrate, with small, protonatable ring 2-carbon substituents being among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S] and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shares low sequence similarity with most structurally characterized members of its family. Structural data show an α-helix-associated histidine side chain that rotates into an Fe (II)-coordinating position following binding of the substrate into an adjacent pocket. TPA interactions with side chains in this pocket were not conserved in homologs with different substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate, the observation that PCA is its oxygenation product, and the close relationship of the TPADO α-subunit to that of anthranilate dioxygenase allowed us to propose a structure-based model for product formation. Future efforts to identify, evolve, or engineer TPADO variants with desirable properties will be enabled by the results described here. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons of diverse forms have found application in different industries and man heavily depends on these compounds for various purposes. Thus, tonnes of thousands of polycyclic aromatic hydrocarbons are released into various water bodies yearly, resulting in pollution with great effects on aquatic lives, man, and the ecosystem at large. Hydrocarbon pollutions in wastewater are remediated by some physical and chemical methods with most of these techniques leaving a different form of harmful byproducts after the remediation. Furthermore, several species of fungi are important in the microbial bioremediation of polycyclic aromatic hydrocarbon in wastewater as they are capable of using these compounds as their source of carbon and energy in the presence of oxygenase. Fungal bioremediation is cost‐effective, safer, and ecologically friendly, in addition to fungi producing polycyclic aromatic hydrocarbon degradative enzymes in high amounts, both intracellularly and extracellularly. Although optimizing the growth requirement of fungi in the field is a major challenge, current advances in the application of fungi in bioremediation address this. This review discusses in detail the technology of fungal bioremediation of polycyclic aromatic hydrocarbon in wastewater and its beneficial roles to man and the ecosystem. The benefits of remediating polycyclic aromatic hydrocarbon‐polluted water with fungi and their metabolites via nanotechnology, immobilization, genomic manipulation, and other technologies to generate value‐added products are highlighted in this manuscript. Information in this review will provide useful important insights to researchers and industrial professionals in the bioremediation of polycyclic aromatic hydrocarbon. [ABSTRACT FROM AUTHOR]

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPScatalyzed production of deoxypodophyllotoxin analogs. [ABSTRACT FROM AUTHOR]

Commonly observed competitive substrate inhibition in cometabolism of organic contaminants is used as rate- and reducing-power-determining factors to develop a kinetic model of the competitive cometabolism. Analogous to the well-known theory of Leudeking-Piret kinetics where the product formation demands reducing power, cometabolism is modeled as a reducing power demanding process that also competes with microbial growth for the available reducing power from the degradation of energy-yielding primary substrate. The model further incorporates other growth-associated phenomena such as substrate inhibition and multiple growth/nongrowth substrate interactions that may occur during cometabolic transformation processes. The kinetic model is used successfully to predict a variety of degradation patterns of growth/nongrowth substrates, displayed by microbial cultures when exposed to different concentration ratios of growth to nongrowth substrate: a complete degradation of nongrowth substrates that coincides with the simultaneous depletion of a growth substrate and, in some other cases, an incomplete degradation of a nongrowth substrate following the complete depletion of a growth substrate. These distinct patterns of substrate degradation are attributed to intrinsic specificities of enzymes for cometabolism and lack of reducing power available from the growth substrate degradation. The efficacy of cometabolic capabilities of actively growing microbial cultures and pre-cultured resting cells is discussed in terms of reducing power available in such systems.

With industrialisation and the rapidly growing agricultural demand, many organic compounds have been leaked into the environment, causing serious damage to the biosphere. Persistent organic pollutants (POPs) are a type of toxic chemicals that are resistant to degradation through normal chemical, biological or photolytic approaches. With their stable chemical structures, POPs can be accumulated in the environment, and transported through wind and water, causing global environmental issues. Many researches have been conducted to remediate POPs contamination using various kinds of biological methods, and significant results have been seen. Microalgae-bacteria consortium is a newly developed concept for biological technology in contamination treatment, with the synergetic effects between microalgae and bacteria, their potential for pollutants degradation can be further released. In this review, two types of POPs (polychlorinated biphenyls and polycyclic aromatic hydrocarbons) are selected as the targeted pollutants to give a systematic analysis of the biodegradation through microalgae and bacteria, including the species selection, the identification of dominant enzymes, as well as the real application performance of the consortia. In the end, some outlooks and suggestions are given to further guide the development of applying microalgae-bacteria consortia in remediating POPs contamination. In general, the coculturing of microalgae and bacteria is a novel and efficient way to fulfil the advanced treatment of POPs in soil or liquid phase, and both monooxygenase and dioxygenase belonging to oxygenase play a vital role in the biodegradation of PCBs and PAHs. This review provides a general guide in the future investigation of biological treatment of POPs. [Display omitted] • Compilation of PCBs and PAHs degradative microalgae, bacteria, and their consortia combinations. • Key enzymes, metabolic pathways and related intermediates in PCBs and PAHs remediation. • Advanced omics techniques are helpful in elucidating degradation processes. • Future perspective in converting after-processing biota to bioresources. [ABSTRACT FROM AUTHOR]

Abstract Background The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate. Results Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases. Conclusion Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

The widespread use of plastics has led to their increasing presence in the environment and subsequent pollution. Some microorganisms degrade plastics in natural ecosystems and the associated metabolic pathways can be studied to understand the degradation mechanisms. Polystyrene (PS) is one of the more recalcitrant plastic polymers that is degraded by only a few bacteria. Exiguobacterium is a genus of Gram-positive poly-extremophilic bacteria known to degrade PS, thus being of biotechnological interest, but its biochemical mechanisms of degradation have not yet been elucidated. Based solely on genome annotation, we initially proposed PS degradation by Exiguobacterium sp. RIT 594 via depolymerization and epoxidation catalyzed by a ring epoxidase. However, Fourier transform infrared (FTIR) spectroscopy analysis revealed an increase of carboxyl and hydroxyl groups with biodegradation, as well as of unconjugated C-C double bonds, both consistent with dearomatization of the styrene ring. This excludes any aerobic pathways involving side chain epoxidation and/or hydroxylation. Subsequent experiments confirmed that molecular oxygen is critical to PS degradation by RIT 594 because degradation ceased under oxygen-deprived conditions. Our studies suggest that styrene breakdown by this bacterium occurs via the sequential action of two enzymes encoded in the genome: an orphan aromatic ring-cleaving dioxygenase and a hydrolase.

Herein, we report a new tripodal tris‐benzimidazole ligand (Tbim) that structurally mimics the 3‐His coordination environment of certain nonheme mononuclear iron oxygenases. The coordination chemistry of Tbim was explored with iron(II) revealing a diverse set of coordination modes. The aerobic oxidation of biomimetic model substrate diethyl‐2‐phenylmalonate was studied using the Tbim−Fe and Fe(OTf)2. [ABSTRACT FROM AUTHOR]

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152Δ matAB that could produce aklavinone, 9- epi -aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152Δ matAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the “MicroBak” preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation.

Globally, neonicotinoid contamination in aquatic environments, including estuarine areas, is a prevailing environmental concern. The estuarine resident marine crustacean sand shrimp Crangon uritai was previously found to have a higher tolerance to neonicotinoids than the marine crustaceans kuruma prawn Penaeus japonicus and mysid Americamysis bahia. Based on these findings, we aimed to explore the mechanisms underlying their differences in insecticide sensitivity. We hypothesized that differences in the structures of their nicotinic acetylcholine receptors (nAChRs) and/or the involvement of a metabolizing enzyme may confer sand shrimp resistance to neonicotinoids. No obvious differences were found in the amino acid residue (position 81) of the loop D region of the nAChR β-subunit among the three crustaceans. A synergistic toxicity bioassay was used to explore candidate metabolizing enzymes, esterase, glutathione S-transferase, and oxygenase. The three species were exposed to two neonicotinoids (acetamiprid and clothianidin) at concentrations equivalent to 1/15–1/10 of 96-h LC50 values and synergists (inhibitors of metabolizing enzyme) or combinations of both. Treatments with the oxygenase inhibitor and the neonicotinoids resulted in increased mortality in sand shrimps but not in kuruma prawns or mysids. Consequently, it was determined that oxygenase may explain the higher resistance of the sand shrimp to neonicotinoid insecticides. [ABSTRACT FROM AUTHOR]

Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the "MicroBak" preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation. [ABSTRACT FROM AUTHOR]

Metagenome from refinery wastewater treatment plant running under nitrogen stress was analyzed for mining of novel aromatic hydrocarbon-degrading bacteria. The sequence data were assembled using metaspade followed by binning using the Metabat tool to assemble genome; where coverage and depth were calculated using bowtie and samtools. The analysis picked a novel genome belonging to family Bradyrhizobiaceae, identified based on 16S rDNA gene which was supported by CheckM and Kraken analysis. Using RAST, the assembled genome showed the capabilities for nitrogen fixation with the utilization of multiple hydrocarbon substrates with 14 different types of oxygenases as mapped by Minpath. An additional genetic feature like genes for stress and resistance towards heavy metals and antibiotic suggested that the genome has gone through the rigorous process of adaptation. If such bacteria could be cultivated then it will open the broad window of bioremediation strategies under nitrogen stress environment. • Genome assembly of a novel nitrogen fixing bacteria from metagenome • Bacterial ability to utilize various aromatic hydrocarbons • Gene cluster for carbon fixation, nitrogen metabolism and urea utilization • Large pool of stress and resistance related genes in the genome [ABSTRACT FROM AUTHOR]

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.

Cells rely on prolyl hydroxylase enzymes to sense low levels of oxygen, but they might act on fewer targets than previously thought.

Rieske oxygenases catalyze an exceptionally broad range of discrete types of reactions despite the utilization of a highly conserved quaternary structure and metal cofactor complement. Oxygen activation within this family occurs at a mononuclear Fe II site, which is located approximately 12 Å from a one-electron reduced Rieske-type iron-sulfur cluster. Electron transfer from the Rieske cluster to the mononuclear iron site occurs during O 2 activation and product formation. A key question is whether all Rieske oxygenase reactions involve the same type of activated oxygen species. This question has been explored using the Rieske oxygenase salicylate 5-hydroxylase, which catalyzes both aromatic hydroxylation of salicylate and aromatic methyl hydroxylation when a methyl substituent is placed in the normal position of aromatic ring hydroxylation. We show here that the combined application of kinetic, biophysical, computational, and isotope effect methods reveals a uniform mechanism for initial O 2 activation and substrate attack for both types of reactivity. However, the mechanism diverges during the later phases of the reactions in response to the electronic nature and geometry of the substrates as well as the lifetime of intermediates. Similar factors may be encountered broadly in the Rieske oxygenase family., (Copyright © 2024. Published by Elsevier Inc.)

The biotechnological potential of Rieske Oxygenases (ROs) and their cognate reductases remains unmet, in part because these systems can be functionally short-lived. Here, we describe a set of experiments aimed at identifying both the functional and structural stability limitations of ROs, using terephthalate (TPA) dioxygenase (from Comamonas strain E6) as a model system. Successful expression and purification of a cofactor-complete, histidine-tagged TPA dioxygenase and reductase protein system requires induction with the Escherichia coli host at stationary phase as well as a chaperone inducing cold-shock and supplementation with additional iron, sulfur, and flavin. The relative stability of the Rieske cluster and mononuclear iron center can then be assessed using spectroscopic and functional measurements following dialysis in an iron chelating buffer. These experiments involve measurements of the overall lifetime of the system via total turnover number using both UV-Visible absorbance and HPLC analyses, as well specific activity as a function of temperature. Important methods for assessing the stability of these multi-cofactor, multi-protein dependent systems at multiple levels of structure (secondary to quaternary) include differential scanning calorimetry, circular dichroism, and metallospectroscopy. Results can be rationalized in terms of three-dimensional structures and bioinformatics. The experiments described here provide a roadmap to a detailed characterization of the limitations of ROs. With a few notable exceptions, these issues are not widely addressed in current literature., (Copyright © 2024. Published by Elsevier Inc.)

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O 2 ) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components., (Copyright © 2024. Published by Elsevier Inc.)

Omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, display a wide range of beneficial effects in humans and animals. Many of the biological functions of PUFAs are mediated via bioactive metabolites produced by fatty acid oxygenases such as cyclooxygenases, lipoxygenases and cytochrome P450 monooxygenases. Liquid chromatography–tandem mass spectrometry-based mediator lipidomics revealed a series of novel bioactive lipid mediators derived from omega-3 PUFAs. Here, we describe recent advances on omega-3 PUFA-derived mediators, mainly focusing on their enzymatic oxygenation pathway, and their biological functions in controlling inflammation and tissue homeostasis. [ABSTRACT FROM AUTHOR]

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O2 via photosynthetic water oxidation. Thus far, research on photosynthesis‐driven bioprocesses mainly focuses on strain development and the proof of principle in small‐scale biocatalytic reaction setups. This study investigates the long‐term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω‐hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity‐independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two‐liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7‐fold increased initial specific activities (5.6 ± 0.1 U/gCDW) and 7.6‐fold increased specific yields on biomass (3.8 ± 0.1 mmolH‐NAME/gCDW) as compared with single aqueous phase biotransformations. Finally, the whole‐cell biotransformation system was successfully scaled from glass tubes to a stirred‐tank photobioreactor. This is the first study reporting the application of the two‐liquid phase concept for efficient phototrophic whole‐cell biocatalysis. [ABSTRACT FROM AUTHOR]

Recently, we observed that at extreme alkaline pH, cytochrome b5 (Cb5) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb5, in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb5 enzymatic activities on the distorted protein before maximum protein unfolding. [ABSTRACT FROM AUTHOR]

Abstract The degradation of trichloroethylene (TCE) and tetrachloroethylene (PCE), in incubations where ammonium was oxidized while iron was being reduced indicates that these compounds can be degraded during the Feammox process by Acidimicrobiaceae sp. A6 (ATCC, PTA-122488). None of these compounds were degraded in incubations to which no ammonium was added, indicating that they were degraded during the oxidation of ammonium. Degradation of TCE and PCE (ranging between 32% and 55%) was observed in incubations with a pure Acidimicrobiaceae sp. A6 culture as well as an Acidimicrobiaceae sp. A6 enrichment culture over a 2-week period. In addition to these batch studies, a column study, with a 5-h hydraulic residence time, was conducted contrasting the degradation of TCE in iron-rich soil columns that were either seeded with a pure or an enrichment culture of Acidimicrobiaceae sp. A6 to achieve ammonium oxidation under iron reduction, and a control column that was initially not seeded and later seeded with Geobacter metallireducens. While there was ∼22% TCE removal in the columns seeded with Acidimicrobiaceae sp. A6, there was no removal in the unseeded column or the column seeded with G. metallireducens which was being operated under iron reducing conditions. Feammox is an anoxic process that requires acidic conditions. Hence, these results indicate that this process might be harnessed where other bioremediation strategies are difficult, since many require neutral or alkaline conditions, and supplying ammonium to an anoxic aquifer is relatively easy, since there are not many processes that will oxidize ammonium in the absence of dissolved oxygen. Graphical abstract Image 1 Highlights • TCE and PCE can be degraded by Acidimicrobiaceae sp. A6 during Feammox. • TCE and PCE were not degraded by Acidimicrobiaceae sp. A6 in the absence of NH 4 +. • TCE/PCE were degraded during Feammox in batch and column experiments. • Production of Fe(II) by itself does not account for the TCE/PCE degradation. [ABSTRACT FROM AUTHOR]