Enzymatic synthesis of indigo derivatives by tuning P450 BM3 peroxygenases

Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.

Vitamin C activation of the biosynthesis of epoxyeicosatrienoic acids

The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+ , and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans- over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxy-genase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.

Formation of (2E)-4-Hydroxy-2-nonenal and (2E)-4-Hydroxy-2-hexenal by Plant Enzymes: A Review Suggests a Role in the Physiology of Plants

It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.

A novel insight of enhancing the hydrogen peroxide tolerance of unspecific peroxygenase from Daldinia caldariorum based on structure

Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of the H_(2)O_(2) tolerance of UPOs is always challenging. In this study, the A161C mutant of r Dca UPO,which originates from Daldinia caldariorum, was found to be highly H_(2)O_(2)-resistant. Compared with the wild type, the mutant r Dca UPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H_(2)O_(2). The crystal structure analysis at 1.47 A showed that r Dca UPOA161C exhibited 10 α-helixes(cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions(Glu86, His87, Asp88 residues). The H_(2)O_(2) tolerance of r Dca UPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, r Dca UPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.

Rhodium complex-anchored and supramolecular polymer-grafted CdS nanoflower for enhanced photosynthesis of H_(2)O_(2) and photobiocatalytic C–H bond oxyfunctionalization

Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS-based semiconductors are promising for the photosynthesis of H_(2)O_(2) owing to their adequately negative potential for oxygen reduction reaction via a proton-coupled electron transfer process,however,they suffer from fast H_(2)O_(2) decomposition on the surface of pristine CdS.Therefore,[Cp*Rh(bpy)H_(2)O]2+,a highly selective proton-coupled electron transfer catalyst,was anchored onto a supramolecular polymer-grafted CdS nanoflower to construct an efficient integrated photocatalyst for generating H_(2)O_(2),mitigating the surface issue of pristine CdS,increasing light absorption,accelerating photonic carrier separation,and enhancing oxygen reduction reaction selectivity to H_(2)O_(2).This photocatalyst promoted the light driven H_(2)O_(2) generation rate up to 1345μmol·L^(-1)·g^(-1)·h^(-1),which was 2.4 times that of pristine CdS.The constructed heterojunction photocatalyst could supply H_(2)O_(2) in situ for nonspecific peroxygenases to catalyze the C–H oxyfunctionalization of ethylbenzene,achieving a yield of 81%and an ee value of 99%under optimum conditions.A wide range of substrates were converted to the corresponding chiral alcohols using this photo-enzyme catalytic system,achieving the corresponding chiral alcohols in good yield(51%–88%)and excellent enantioselectivity(90%–99%ee).