A Permissive Amide <i>N</i>‑Methyltransferase for Dithiolopyrrolones

Amide N-methylation is important for the activity and permeability of bioactive compounds but can be challenging to perform selectively. The broad-spectrum antimicrobial natural products thiolutin and holomycin differ only by an N-methyl group at the endocyclic amide of thiolutin, but only thiolutin exhibits antifungal activity. The enzyme responsible for amide N-methylation in thiolutin biosynthesis has remained elusive. Here, we identified and characterized the amide N-methyltransferase DtpM that is encoded >400 kb outside of the thiolutin gene cluster. DtpM catalyzes efficient conversion of holomycin to thiolutin, exhibits broad substrate scope toward dithiolopyrrolones, and has high thermal stability. In addition, sequence similarity network analysis suggests DtpM is more closely related to phenol O-methyltransferases than some amide methyltransferases. This study expands the limited examples of amide N-methyltransferases and may facilitate chemoenzymatic synthesis of diverse dithiolopyrrolone compounds as potential therapeutics

Chemical and Enzymatic Transformations of Nimesulide to GSH Conjugates through Reductive and Oxidative Mechanisms

Nimesulide (NIM) is a nonsteroidal anti-inflammatory drug, and clinical treatment with NIM has been associated with severe hepatotoxicity. The bioactivation of nitro-reduced NIM (NIM-NH₂), a major NIM metabolite, has been thought to be responsible for the hepatotoxicity of NIM. However, we found that NIM-NH₂ did not induce toxic effects in primary rat hepatocytes. This study aimed to investigate other bioactivation pathways of NIM and evaluate their association with hepatotoxicity. After incubating NIM with NADPH- and GSH-supplemented human or rat liver microsomes, we identified two types of GSH conjugates: one was derived from the attachment of GSH to NIM-NH₂ (NIM-NH₂-GSH) and the other one was derived from a quinone-imine intermediate (NIM-OH-GSH). NIM-NH₂-GSH was generated not only by the oxidative activation of NIM-NH₂ but also from the reductive activation of NIM. Both NADPH and GSH could act as reducing agents. Moreover, aldehyde oxidase also participated in the reductive activation of NIM. NIM-OH-GSH was generated mainly from NIM via epoxidation with CYP1A2 as the main catalyzing enzyme. NIM was toxic to both primary human and rat hepatocytes, with IC₅₀ values of 213 and 40 μM, respectively. Inhibition of the oxidative and reductive activation of NIM by the nonspecific CYP inhibitor 1-aminobenzotriazole and selective aldehyde oxidase inhibitor estradiol did not protect the cells from NIM-mediated toxicity. Moreover, pretreating cells with l-buthionine-sulfoximine (a GSH depletor) did not affect the cytotoxicity of NIM. These results suggested that oxidative and reductive activation of NIM did not cause the hepatotoxicity and that the parent drug concentration was associated with the cytotoxicity

DataSheet_1_The effects of atmospheric nitrogen deposition in coral-algal phase shifts on remote coral reefs.pdf

Remote seawater has been considered a potential refuge for corals in the face of anthropogenic disturbances. However, these remote areas may receive increased atmospheric N deposition, and the ecological consequences remain unclear. This field survey revealed coral-algal phase shifts in the mid-north of the South China Sea. These shifts were observed in 44%, 13.6%, and 26.5% of the sampled reef sites at depths of 1-4 m, 5-8 m, and 10-15 m, respectively. Over 50% of sections in the deeper depths hosted fewer corals compared to shallower areas, coinciding with a higher abundance of macroalgae in the deeper layers. Furthermore, based on long-term observation of atmospheric N flux, laboratory experiments were conducted to explore the cause of coral declines. The results indicate that N supply efficiently promoted macroalgae growth. The saturation of N absorption by macroalgae occurred within 2 weeks, leading to nutrient accumulation in seawater, especially nitrate, which had a direct impact on corals. While moderate N fluxes appeared to mitigate coral bleaching, high N fluxes, even with a balanced P supply or medium level of nutrients with an imbalanced N/P ratio, can both increase the susceptibility of corals to heat bleaching. This study explains the coral-algal phase shift in remote and relatively deep seawater and improves understanding of the cause-and-effect relationship between atmospheric N deposition and coral reef decline.</p

Characteristics of Cu-NPs used in this study.

<p>Transmission electron microscopy (TEM) image, scale bar = 100 nm; (B) size distribution of Cu-NPs, with measurements obtained from the TEM images.</p

Reactive oxygen species (ROS) formation (A)and oxidative stress(B-E) in the primary hepatocytes of juvenile <i>E</i>.<i>coioides</i> after CuSO₄ or Cu-NPs exposure.

<p>Data are means ±SD (n = 3). Significant differences (<i>p <</i>0.05) among treatments were indicated by different letters.</p

Cell viability and lactate dehydrogenase (LDH) leakage in primary hepatocytes of juvenile <i>E</i>.<i>coioides</i> after CuSO₄ or Cu-NPs exposure.

<p>Data are means ±SD (n = 3). Significant differences (<i>p <</i>0.05) among treatments were indicated by different letters.</p

p53 protein expression in primary hepatocytes of juvenile <i>E</i>.<i>coioides</i> after CuSO₄ or Cu-NPs exposure.

<p>p53 protein expression in primary hepatocytes of juvenile <i>E</i>.<i>coioides</i> after CuSO₄ or Cu-NPs exposure.</p

Scheme showing the proposed mechanisms of Cu-NPs and CuSO₄ toxicity to primary hepatocytes of <i>E</i>.<i>coioides</i>.

<p>Cu-NPs and CuSO₄ exhibited similar types of toxic mechanisms.</p