Related Experiment Video
Updated: Jun 16, 2026
08:57
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
A Single Amino Acid Substitution Reprograms ROS Selectivity and Catalytic Function in DyP Peroxidases
Ulises A Zitare1,2, María A Castro1,2, Magalí F Scocozza1,2
1Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina.
Inorganic Chemistry
|June 15, 2026
Summary
Dye-decolorizing peroxidases (DyPs) are activated by reactive oxygen species (ROS) through dynamic gating. Protein engineering can tune ROS selectivity, enhancing biotechnological applications.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Dye-decolorizing peroxidases (DyPs) are heme enzymes with significant biotechnological potential.
- Understanding the structural basis for DyP activation by reactive oxygen species (ROS) is crucial for their application.
Purpose of the Study:
- To investigate the mechanism of ROS activation in class I DyPs.
- To elucidate the structure-function relationship governing ROS selectivity in DyPs.
- To explore protein engineering strategies for tuning DyP ROS preferences.
Main Methods:
- Comparative study of *Bacillus subtilis* DyP (BsDyP) and *Thermobifida fusca* DyP (TfuDyP), including a BsDyP N244L variant.
- Enzyme kinetics, ROS-selective electroreductive activation assays.
- Spectroscopic techniques (UV-vis, resonance Raman), X-ray crystallography, and molecular dynamics simulations.
Main Results:
- Identified dynamic ROS gating as a key mechanism controlling DyP activation and efficiency.
- BsDyP WT is preferentially activated by hydroxyl radicals (•OH), while TfuDyP primarily uses hydrogen peroxide (H2O2).
- The BsDyP N244L mutation shifted its ROS usage and catalytic efficiency towards TfuDyP, demonstrating the impact of specific substitutions.
Conclusions:
- Distal heme pocket organization and access tunnel architecture dictate ROS selectivity in DyPs.
- ROS gating is the primary determinant of DyP activation.
- Protein engineering offers a viable approach to modulate ROS selectivity in DyPs for tailored applications.
Related Concept Videos
02:04Oxidation of Alkenes: Anti Dihydroxylation with Peroxy Acids
Diols are compounds with two hydroxyl groups. In addition to syn dihydroxylation, diols can also be synthesized through the process of anti dihydroxylation. The process involves treating an alkene with a peroxycarboxylic acid to form an epoxide. Epoxides are highly strained three-membered rings with oxygen and two carbons occupying the corners of an equilateral triangle. This step is followed by ring-opening of the epoxide in the presence of an aqueous acid to give a trans diol.
01:27Protein Import into the Peroxisomes
Cells contain membrane-bound organelles called peroxisomes that oxidize organic molecules by transferring hydrogen atoms to oxygen, producing hydrogen peroxide. Peroxisomes enzymatically convert the released hydrogen peroxide into water and oxygen.
Peroxisomal Protein Import:
Peroxisomes lack the genetic machinery required to code for their own proteins. Hence, most peroxisomal membrane, lumenal and transmembrane proteins are synthesized in the cytoplasm or ER and transported to the peroxisome...
Peroxisomal Protein Import:
Peroxisomes lack the genetic machinery required to code for their own proteins. Hence, most peroxisomal membrane, lumenal and transmembrane proteins are synthesized in the cytoplasm or ER and transported to the peroxisome...
02:44Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide
Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double bond with two different stereochemical approaches, namely anti and syn. Dihydroxylation using osmium tetroxide progresses with syn stereochemistry.
02:35Regioselectivity of Electrophilic Additions-Peroxide Effect
In the presence of organic peroxides, the addition of hydrogen bromide to an alkene yields the isomer that is not predicted by Markovnikov’s rule. For example, the addition of hydrogen bromide to 2-methylpropene in the presence of peroxides gives 1-bromo-2-methylpropane. This addition reaction proceeds via a free radical mechanism, which reverses the regioselectivity. The free radical reaction mechanism involves three stages: initiation, propagation, and termination.
01:24Peroxisomes
Peroxisomes are specialized organelles present in fungi, plant, and animal cells. It can vary in number, size, morphology, and activity depending on the type of tissue and the nutritional state of the cell. For example, cells with active lipid metabolism, such as adipocytes, neurons, and hepatocytes, have more peroxisomes than other cells in the body. Besides their primary role in breaking down complex organic molecules, peroxisomes can also synthesize specific macromolecules and participate in...
01:26Protein Modifications in the RER
Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.