Peroxyzymes simply use H 2 O 2 as a cosubstrate to oxidize a broad range of inert C–H bonds. The lability of many peroxyzymes against H 2 O 2 can be addressed by a controlled supply of H 2 O 2 , ideally in situ. Here, we report a simple, robust, and water-soluble anthraquinone sulfonate (SAS) as a promising organophotocatalyst to drive both haloperoxidase-catalyzed halogenation and peroxygenase-catalyzed oxyfunctionalization reactions. Simple alcohols, methanol in particular, can be used both as a cosolvent and an electron donor for H 2 O 2 generation. Very promising turnover numbers for the biocatalysts of up to 318 000 have been achieved.

The heme enzyme chlorite dismutase (Cld) catalyzes O–O bond formation as part of the conversion of the toxic chlorite (ClO 2 – ) to chloride (Cl – ) and molecular oxygen (O 2 ). Enzymatic O–O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae ( Ao Cld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. Ao Cld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, Ao Cld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at k obs = 2–5 × 10 4 s –1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of Ao Cld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety’s reorganization and thereby maximize the enzyme’s catalytic efficiency.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages.

The oxidation of alkanes into valuable chemical products is a vital reaction in organic synthesis. This reaction, however, is challenging, owing to the inertness of C−H bonds. Transition metal catalysts for C−H functionalization are frequently explored. Despite chemical alternatives, nature has also evolved powerful oxidative enzymes (e. g., methane monooxygenases, cytochrome P450 oxygenases, peroxygenases) that are capable of transforming C−H bonds under very mild conditions, with only the use of molecular oxygen or hydrogen peroxide as electron acceptors. Although progress in alkane oxidation has been reviewed extensively, little attention has been paid to small alkane oxidation. The latter holds great potential for the manufacture of chemicals. This Minireview provides a concise overview of the most relevant enzyme classes capable of small alkanes (C<6) oxyfunctionalization, describes the essentials of the catalytic mechanisms, and critically outlines the current state‐of‐the‐art in preparative applications.

Excess amounts of uric acid in humans leads to hyperuricemia, which is a biochemical precursor of gout and is also associated with various other disorders. Gout is termed as crystallization of uric acid, predominantly within joints. The burden of hyperuricemia and gout has increased worldwide due to lifestyle changes, obesity, and consumption of purine-rich foods, fructose-containing drinks, and alcoholic beverages. Some of the therapies available to cure gout are associated with unwanted side-effects and antigenicity. We propose an attractive and safe strategy to reduce purine content in beverages using enzymatic application of purine degrading enzymes such as adenine deaminase (ADA) and guanine deaminase (GDA) that convert adenine and guanine into hypoxanthine and xanthine, respectively. We cloned, expressed, purified, and biochemically characterized both adenine deaminase (ADA) and guanine deaminase (GDA) enzymes that play important roles in the purine degradation pathway of Kluyveromyces lactis, and demonstrate their application in lowering purine content in a beverage. The popular beverage beer has been selected as an experimental sample as it confers higher risks of hyperuricemia and gout. Quantification of purine content in 16 different beers from the Indian market showed varying concentrations of different purines. Enzymatic treatment of beer samples with ADA and GDA showed a reduction of adenine and guanine content, respectively. These enzymes in combination with other purine degrading enzymes showed marked reduction in purine content in beer samples. Both enzymes can work at 5.0–8.0 pH range and retain >50% activity at 40°C, making them good candidates for industrial applications.

Chlorite dismutase is a unique heme enzyme that catalyzes the conversion of chlorite to chloride and molecular oxygen. The enzyme is highly specific for chlorite but has been known to bind several anionic and neutral ligands to the heme iron. In a pH study, the enzyme changed color from red to green in acetate buffer pH 5.0. The cause of this color change was uncovered using UV-visible and EPR spectroscopy. Chlorite dismutase in the presence of acetate showed a change of the UV-visible spectrum: a redshift and hyperchromicity of the Soret band from 391 to 404 nm and a blueshift of the charge transfer band CT1 from 647 to 626 nm. Equilibrium binding titrations with acetate resulted in a dissociation constant of circa 20 mM at pH 5.0 and 5.8. EPR spectroscopy showed that the acetate bound form of the enzyme remained high spin S = 5/2, however with an apparent change of the rhombicity and line broadening of the spectrum. Mutagenesis of the proximal arginine Arg183 to alanine resulted in the loss of the ability to bind acetate. Acetate was discovered as a novel ligand to chlorite dismutase, with evidence of direct binding to the heme iron. The green color is caused by a blueshift of the CT1 band that is characteristic of the high spin ferric state of the enzyme. Any weak field ligand that binds directly to the heme center may show the red to green color change, as was indeed the case for fluoride.

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2− (chlorite) to innocuous Cl− and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

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