Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of L-amino acids at ,  and  positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs -elimination of O-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C-C bond in 3 and complete the -substitution reaction. CndF displays substrate promiscuity towards different -keto carboxylate and esters. Using a recombinant Aspergillus strain expressing CndF and CndE, feeding various alkyl--keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.Nature is remarkable in building and using structurally diverse amino acids. 1-2 Nonproteinogeneic amino acids (NAAs), which constitute 96% of the naturally occurring amino acids, 1 are frequently incorporated into small molecules to broaden reactivity and to establish biologically relevant conformers. 3 Peptides that contain NAAs are less susceptible to proteolysis, thereby increasing the halflives during circulation. [4][5] The usefulness of NAAs have also been extensively explored through incorporation into recombinant proteins in Escherichia coli, yeast, and mammalian cells. 6 Because of these

Aromatic L-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze Ltryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative L-tryptophandecarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis (PcncAAAD) that exhibits substrate permissiveness toward L-phenylalanine, L-tyrosine, and L-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-β-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.

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Bitter gourd (Momordica charantia L.) is a common vegetable grown widely in Asia that is used as a traditional medicine. The objective of this study was to investigate whether wild bitter gourd possessed protective effects against chronic alcohol-induced liver injury in mice. C57BL/6 mice were fed an alcohol-containing liquid diet for 4 weeks to induce alcoholic fatty liver. Meanwhile, mice were treated with ethanol extracts from four different wild bitter gourd cultivars: Hualien No. 1', Hualien No. 2', Hualien No. 3' and Hualien No. 4'. The results indicated that the daily administration of 500 mg kg body weight(-1) of a Hualien No. 3' extract (H3E) or a Hualien No. 4' extract (H4E) markedly reduced the steatotic alternation of liver histopathology. In addition, the activation of serum aminotransferases (AST and ALT) and the accumulation of hepatic TG content caused by alcohol were ameliorated. The hepatoprotective effects of H3E and H4E involved the enhancement of the antioxidant defence system (GSH, GPx, GRd, CAT and SOD), inhibition of lipid peroxidation (MDA) and reduction of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the liver. Moreover, H3E and H4E supplementation suppressed the alcohol-induced elevation of CYP2E1, SREBP-1, FAS and ACC protein expression. These results demonstrated that ethanol extracts of Hualien No. 3' and Hualien No. 4' have beneficial effects against alcoholic fatty liver, in which they attenuate oxidative stress and inflammatory responses.

Purpose: To compare survival and histologic features of hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome) cases to well-matched sporadic colon cancers from the same patient population. Experimental Design: Between January 1995 and March 2002, a total of 5,138 consecutive patients underwent resection of primary colorectal adenocarcinoma in a single institution. According to the Amsterdam criteria, 56 HNPCC patients were matched to 147 sporadic colorectal cancer (SCRC) with no family history of cancer and with the same gender, tumor location, and age within 3 years. Immunohistochemical analyses were done for MUC1, MUC2, MUC3, and MUC5AC. Results: The HNPCC group had a marginally significantly better long-term outcome than the SCRC group (P = 0.058). The trend disappeared after adjustment by tumor-node-metastasis stage in a Cox model (P = 0.774). We noted a difference of >50% in the 5-year cancer-specific survival rates of HNPCC-and SCRC-mucinous groups (92% versus 31%, P = 0.0003). Interaction between mucin and HNPCC and its effects on survival were further confirmed by comparing the Cox models with and without interaction terms (hazard ratio, 0.1; P = 0.034 with adjusting stage). Patients with tumors showing dual expression of mucin and MUC1, which appeared in 11% of those with HNPCC and 50% of those with SCRC, had a lower 5-year cancer-specific survival rate than patients without (30% versus 60%; P = 0.004 by log-rank test; P = 0.039 with adjustment for tumor-node-metastasis stage). Conclusions: These results suggest that mucin has an inverse effect on survival in patients with HNPCC and SCRC, which might be partly explained by a lower prevalence of MUC1expression in the mucinous HNPCC group than in the SCRC groups.

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The intestinal microbiome plays an important role in the pathogenesis of liver diseases. Alcohol intake induces gut microbiota dysbiosis and alters its function. This study investigated the antibiotic effect of allicin in mice with hepatic steatosis. Male C57BL/6 mice were administered an ethanol diet supplemented with allicin (5 and 20 mg/(kg bw day)) for 4 weeks. Allicin modified the gut microbiota composition. Cecal microbiota exhibited a positive correlation with alcohol and hepatic triacylglycerol, but were suppressed with allicin. Ethanol diet with 5 mg of allicin induced a lower intestinal permeability compared to the ethanol diet alone. Allicin mediated the lipopolysaccharide (LPS)–CD14–toll-like receptor 4 (TLR4)-induced hepatic inflammation pathway by reducing LPS, CD14, TLR4, and pro-inflammatory cytokinestumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. However, hepatic inflammation primarily resulted from alcohol toxicity rather than LPS production in the gut. The prediction of functional profiles from metagenomic 16S ribosomal RNA (rRNA) data revealed different functional profiles in each group. The predicted aldehyde dehydrogenase tended to increase in alcoholic mice administered allicin. The predicted LPS-related pathway and LPS biosynthesis protein results exhibited a similar trend as plasma LPS levels. Thus, alcohol and allicin intake shapes the gut microbiota and its functional profile and improves the CD14–TLR4 pathway to alleviate inflammation in the liver.

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