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1. Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

   https://doi.org/10.1021/acscatal.1c04770  

   Traore, Ephrahime S.; Liu, Aimin (April 2022, ACS Catalysis)

   Pimchai Chaiyen (Ed.)

   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. 

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2. Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities

   https://doi.org/10.1074/jbc.RA118.002698  

   Yang, Yu; Liu, Fange; Liu, Aimin (January 2018, Journal of Biological Chemistry)

   3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O2 and inserts both O atoms into 3- hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O2, even though local O2 concentrations and diffusion rates are relatively low. Here, a close inspection of the HAO structures revealed that substrate- and inhibitor-bound structures exhibit a closed conformation with three hydrophobic loop regions moving toward the catalytic iron center, whereas the ligand-free structure is open. We hypothesized that these loop movements enhance O2 binding to the binary complex of HAO and to 3-HAA. We found that the carboxyl end of 3-HAA triggers the changes in two loop regions and that the third loop movement appears to be driven by an H-bond interaction between Asn-27 and Ile-142. Mutational analyses revealed that N27A, I142A, and I142P variants cannot form a closed conformation, and steady-state kinetic assays indicated that these variants have a substantially higher Km for O2 than wild-type HAO. This observation suggested enhanced hydrophobicity at the iron center resulting from the concerted loop movements after the binding of the primary substrate, which is hydrophilic. Given that O2 is nonpolar, the increased hydrophobicity at the Fe center of the complex appears to be essential for rapid O2 binding and activation, explaining the reason for the 3-HAA–induced loop movements. As substrate binding–induced open-to-closed conformational changes are common, the results reported here may help further our understanding of how oxygen is enriched in the nonheme Fe-dependent dioxygenases. 

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3. Characterization of the substrate specificity and regioselectivity of ring-cleavage of Pseudomonas putida DLL-E4 hydroquinone 1,2-dioxygenase (PnpC1C2)

   https://doi.org/10.1007/s00775-025-02101-4  

   Machonkin, Timothy E; Maker, Madeleine S; Ganjoloo, Nandin; Conkin, Drew F (February 2025, JBIC Journal of Biological Inorganic Chemistry)

   PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into -hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear nonheme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydro-quinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher , and chloro- and bromohydroquinone showed a moderately lower , but all had a within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure-function correlations among the hydroquinone ring-cleaving dioxygenases. 

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4. Carbon–fluorine bond cleavage mediated by metalloenzymes

   https://doi.org/10.1039/C9CS00740G  

   Wang, Yifan; Liu, Aimin (January 2020, Chemical Society Reviews)

   Fluorochemicals are a widely distributed class of compounds and have been utilized across a wide range of industries for decades. Given the environmental toxicity and adverse health threats of some fluorochemicals, the development of new methods for their decomposition is significant to public health. However, the carbon–fluorine (C–F) bond is among the most chemically robust bonds; consequently, the degradation of fluorinated hydrocarbons is exceptionally difficult. Here, metalloenzymes that catalyze the cleavage of this chemically challenging bond are reviewed. These enzymes include histidine-ligated heme-dependent dehaloperoxidase and tyrosine hydroxylase, thiolate-ligated heme-dependent cytochrome P450, and four nonheme oxygenases, namely, tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, 2-oxoglutarate-dependent hydroxylase, Rieske dioxygenase, and thiol dioxygenase. While much of the literature regarding the aforementioned enzymes highlights their ability to catalyze C–H bond activation and functionalization, in many cases, the C–F bond cleavage has been shown to occur on fluorinated substrates. A copper-dependent laccase-mediated system representing an unnatural radical defluorination approach is also described. Detailed discussions on the structure–function relationships and catalytic mechanisms provide insights into biocatalytic defluorination, which may inspire drug design considerations and environmental remediation of halogenated contaminants. 

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5. A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins

   https://doi.org/10.1002/adsc.202400656  

   Rutkowski, Bailey_N; Talley, Ella_R; Combs, Jasney; Froese, Jordan_T (September 2024, Advanced Synthesis & Catalysis)

   Abstract Rieske dioxygenase enzymes can perform thecis‐dihydroxylation of aliphatic olefins, representing a potential green alternative to established methods of performing this important transformation. However, the activity of the natural enzymes in this context is low relative to their more well‐known activity in thecis‐dihydroxylation of aromatics. To enable the engineering of dioxygenase enzymes for improved activity in the dihydroxylation of aliphatic olefins, we have developed an assay system to detect the relevant diol metabolites produced from whole‐cell fermentation cultures. Optimization studies were carried out to maximize the sensitivity of the assay system, and its utility in thein vitroscreening of enzyme variant libraries was demonstrated. The assay system was utilized in screening studies that identified Rieske dioxygenase variants with significantly improved activity in the dihydroxylation of aliphatic olefins relative to the wild‐type enzyme. 

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