Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis.

Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Hunteriasines A - D, tryptamine-derived alkaloids from Hunteria umbellata.

Four undescribed tryptamine-derived alkaloids, hunteriasines A - D, were isolated and identified from Hunteria umbellata (Apocynaceae), together with fifteen known indole alkaloids. The chemical structure and absolute configuration of hunteriasine A were determined by spectroscopic and X-ray crystallographic data analyses. Hunteriasine A, featuring with a unique scaffold comprised of tryptamine and an unprecedented "12-carbon unit" moiety, is a zwitterionic indole-derived and pyridinium-containing alkaloid. Hunteriasines B - D were identified by spectroscopic data analyses and theoretical calculations. A plausible biogenetic pathway for hunteriasines A and B was proposed. The lipopolysaccharide-stimulated mouse macrophage cell line J774A.1 cell-based bioactivity assays revealed that (+)-eburnamine, strictosidinic acid, and (S)-decarbomethoxydihydrogambirtannine enhance the release of interleukin-1ß.

Biosensor-Enabled Discovery of CaERG6 Inhibitors and Their Antifungal Mode of Action against Candida albicans.

Fungal infections caused by opportunistic pathogens, such as Candida albicans, are generally underappreciated by the public in spite of their high mortality rates. Antifungal arsenals are extremely limited. Herein, based on biosynthetic pathway comparison and functional characterization, CaERG6, a crucial sterol 24-C-methyltransferase involved in the biosynthesis of ubiquitous ergosterol in C. albicans, was set up as an antifungal target. CaERG6 inhibitors were identified from the in-house small-molecule library by a biosensor-based high-throughput screening. The CaERG6 inhibitor NP256 (palustrisoic acid E) is a potential antifungal natural product that acts by inhibiting ergosterol biosynthesis, downregulating the gene expression level in hyphal formation, blocking biofilm formation, and disrupting morphological transition in C. albicans. NP256 enhances C. albicans susceptibility to some known antifungals significantly. The present study demonstrated the CaERG6 inhibitor NP256 as a potential class of antifungal compound for monotherapy or combinatory therapy.

Antimicrobial glycoalkaloids from the tubers of Stephania succifera.

Three new glycoalkaloids, N-formyl-asimilobine-2-O-ß-D-glucoside (1), (-)-1-O-ß-D-glucoside-8-oxotetrahydropalmatine (2), and 1-N-monomethylcarbamate-argentinine-3-O-ß-D-glucoside (3) were isolated from tubers of Stephania succifera. The structures were established based on spectroscopic analysis, and the antimicrobial activities of the three glycoalkaloids are reported.

A new antibacterial denitroaristolochic acid from the tubers of Stephania succifera.

A new denitroaristolochic acid, demethylaristofolin C (1), together with six known alkaloids, crebanine N-oxide (2), (-)-sukhodianine-ß-N-oxide (3), palmatine (4), corydalmine (5), dehydrocorydalmine (6), and corynoxidine (7), was isolated from the tubers of Stephania succifera. The structure of demethylaristofolin C was elucidated by spectroscopic techniques (UV, IR, 1D, and 2D NMR) and HR-ESI-MS analyses. These compounds exhibited antibacterial activities against Staphylococcus aureus and methicillin-resistant S. aureus strains in different degrees.

A new prenylated xanthone from the branches of Calophyllum inophyllum.

The investigation of chemical constituents from the branches of Calophyllum inophyllum Linn led to the isolation of a new prenylated xanthone, named caloxanthone Q (1), together with three known compounds, 2-deprenylrheediaxanthone B (2), jacareubin (3), and 6-deoxyjacareubin (4). Their structures were completely elucidated on the basis of spectroscopic methods (UV, IR, HR-ESI-MS, 1D NMR, and 2D NMR).

Enzyme-assisted aqueous extraction of oleosomes from soybeans (Glycine max).

Oleosomes, with their unique structural proteins, the oleosins, are known to be useful in cosmetics and other emulsion applications. A procedure to fractionate intact oleosomes to produce soybean oil without the use of organic solvents was investigated. Process parameters, enzyme treatment, filtration, cell lysis, and centrifugation, were studied. Successive extractions of the residue, eliminating the filtration step, pressurization, or ultrasonication of soybean flour prior to enzyme treatment and enzyme treatment on the residue, were the key steps. A mixture of Multifect Pectinase FE, Cellulase A, and Multifect CX 13L in equal proportion gave 36.42-63.23% of the total soybean oil from oleosomes, respectively, for 45 and 180 s of blending time, compared to the conventional method with lower yields (34.24 and 28.65%, respectively, for 45 and 180 s of blending time). Three successive extractions of the residue increased the oil yield to a maximum of 84.65% of the total soybean oil recovered in intact oleosomes. The percentage of lipid in the supernatant fraction decreased to a minimum value of 0.33% with the use of the enzymes at a 3% dosage. The results are considered to be useful for developing large-scale and efficient extraction of oleosomes from soybean.