Metabolites with antioxidant and α-glucosidase inhibitory activities produced by the endophytic fungi Aspergillus niger from Pachysandra terminalis.

One new compound and 13 known compounds were isolated from Aspergillus niger, a plant endophytic fungus of Pachysandra terminalis collected from Qinling Mountains, Xi'an, China. The structure of new compound 1 was classically determined by extensive spectroscopic analysis. Compounds 5, 6, 8, and 14 were first reported from Aspergillus, while compound 2 was isolated from A. niger for the first time. All isolated compounds were further evaluated for their antioxidant and α-glucosidase inhibitory activities. Compounds 2 and 3 exhibited significant antioxidant activities with IC50 values of 31.64 µm and 24.32 µm, respectively, similar to the positive control ascorbic acid. Additionally, compound 1 displayed remarkable inhibitory activity against α-glucosidase with an IC50 value of 96.25 µm, which was 3.4-fold more potent than that of the positive control acarbose. Compound 1 has great potential for development as a new lead compound owing to its simple structure and remarkable biological activity.

Liquid chromatography/mass spectrometry analysis of branched fructans produced in vitro with 13C-labeled substrates.

RATIONALE: Fructans are carbohydrates predominantly based on fructose which are generally considered to be soluble dietary fibers with health-promoting properties. It is known that the nutritional properties of fructans are affected by their structure. This study focused on structural determination of branched fructans, as the most important dietary fructans are branched graminan-type fructans. METHODS: Branched fructans were synthesized enzymatically by incubation of a heterologously expressed sucrose:fructan 6-fructosyltransferase (6-SFT) from Pachysandra terminalis with native or (13)C-labeled substrates. Liquid chromatography/mass spectrometry (LC/MS) was used for the structural identification of branched fructans. The MS(2) fragmentation of these compounds is described for the first time. Analytes were charged by electrospray ionization in negative mode and a quadrupole mass analyzer was used for MS(2) analysis. RESULTS: The MS(2) fragmentation patterns of branched and linear fructans were shown to differ and distinctive ion formation allowed differentiation between all branched fructan isomers formed. P. terminalis 6-SFT preferred extending the existing fructan branch rather than creating a new branch. CONCLUSIONS: The MS(2) fragmentation patterns described in the current paper now allow rapid screening of large sample sets for the presence of branched, graminan-type fructans. Furthermore, the data enables the characterization of fructan-metabolizing enzymes by identification of the fructan structures produced by in vitro reactions as described here for P. terminalis 6-SFT.

Crystal structure of 6-SST/6-SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6-kestose.

Fructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan- and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a ß(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a ß(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.