Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d -ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.     

Dose-dependent effect in the inhibition of oxidative stress and anticlastogenic potential of ginger in STZ induced diabetic rats

Ginger is an important medicinal herb has numerous bioactive components and is used in the management, control and/or treatment of diseases including diabetes mellitus. The present study was undertaken to see the dose–response effect of ginger and evaluate the possible protective effects of dietary ginger on oxidative stress and genotoxicity induced by streptozotocin (STZ) diabetic rats. Inbred male Wistar/NIN rats of 8–9 weeks old were treated with 30 mg/kg of STZ. Rats were divided into different groups of control, diabetic non-treated, and diabetic treated with ginger powder at 0.5%, 1% and 5% respectively. After feeding for a month, blood and tissues were collected to see the effect of ginger on antioxidant status, DNA damage and bone marrow genotoxicity. In this study ginger exerted a protective effect against STZ-induced diabetes by modulating antioxidant enzymes and glutathione and down regulating lipid and protein oxidation and inhibition in genotoxicity in a dose–response manner.     

Optimising by the response surface methodology the enzymatic elimination of clogging of a microfiltration membrane by pectin cake

The aim of this paper, in which clogging occurs on the filter at a constant vacuum pressure and constant pectin weight, is to find the optimal values of the variables, temperature and time, to know the best conditions to reduce this clogging by enzymatic hydrolysis. Moreover, different properties of the compression of the cake in the filter were determined such as the filtering medium total resistance (R_fT), bed porosity (ε) and the cake‐specific resistance (α) at different times and temperatures. The filter area was 0.142 m², and the value of the mass fraction of solids in the filtered suspension (S) was 0.12. Scanning electron microsopy (SEM) analyses were performed. The results showed that the compressive stress corresponding to the cake improves the flow rate in the filtration flux at the optimal temperature and time for the process, where the enzyme reaches its highest activity. The test results of this study can be applied in different system of filters during cross‐flow filtration.     

Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes

The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Herein, we report functional synthetic, fused FDH-FR proteins containing various FDHs and FRs joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in Escherichia coli.     

Copper(I)-Dioxygen Adducts and Copper Enzyme Mechanisms

Primary copper(I)-dioxygen (O₂) adducts, cupric-superoxide complexes, have been proposed intermediates in copper-containing dioxygen-activating monooxygenase and oxidase enzymes. Here, mechanisms of C−H activation by reactive copper-(di)oxygen intermediates are discussed, with an emphasis on cupric-superoxide species. Over the past 25 years, many synthetically derived cupric-superoxide model complexes have been reported. Due to the thermal instability of these intermediates, early studies focused on increasing their stability and obtaining physical characterization. More recently, in an effort to gain insight into the possible substrate oxidation step in some copper monooxygenases, several cupric-superoxide complexes have been used as surrogates to probe substrate scope and reaction mechanisms. These cupric superoxides are capable of oxidizing substrates containing weak O−H and C−H bonds. Mechanistic studies for some enzymes and model systems have supported an initial hydrogen-atom abstraction via the cupric-superoxide complex as the first step of substrate oxidation.     

Activity of Fluorine‐Containing Analogues of WC‐9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine‐containing derivatives, the 3‐(3‐fluorophenoxy)‐ and 3‐(4‐fluorophenoxy)phenoxyethyl thiocyanates, exhibited half‐maximal effective concentration (EC₅₀) values of 1.6 and 4.9 μm , respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC‐9 against intracellular T. cruzi (EC₅₀ values of 5.4 and 5.7 μm , respectively). In addition, 2‐[3‐ (phenoxy)phenoxyethylthio]ethyl‐1,1‐bisphosphonate, which is a hybrid inhibitor containing 3‐phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub‐micromolar levels (EC₅₀=0.7 μm ), which suggests a combined inhibitory effect of the two functional groups.