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1. 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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2. 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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3. 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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4. 6-substituted derivatives of dopamine as substrates of L-DOPA dioxygenase: Understanding steric and electronic substituent effects

   https://doi.org/10.1021/scimeetings.1c01136  

   David Strzeminski, Alexander M. (August 2021, Directory of the American Chemical Society)

   Dioxygenase enzymes are nature’s catalysts for the breakdown of catecholic rings, such as those found in the woody tissue of plants. This chemistry has tremendous potential not only in the degradation of plant material, but also in natural product biosynthesis. A suite of synthetic dopamines, derivatized at the 6-position and varied in substituent size and electron-withdrawing character, were examined as substrates of S. lincolnensis L-DOPA dioxygenase via in-silico docking and subsequent analysis of the enzyme catalyzed transformations by mass spectrometry and UV-Visible spectroscopy. 6-bromodopamine, 6-cyanodopamine and 6-carboxydopamine were robust substrates, while cyclicdopamine and 6-nitrodopamine exhibited limited turnover. Docking and kinetic experiments are used to explain these substrate preferences by L-DOPA dioxygenase. 

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5. Investigation of the Effects of Mutating Iron-Coordinating Residues in Rieske Dioxygenases

   https://doi.org/10.33043/FF.10.1.90-108  

   Froese, Jordan; Betts, Phillip (May 2024, Fine Focus)

   Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. In an attempt to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity. 

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