Structural analysis and reaction mechanism of the disproportionating enzyme (D-enzyme) from potato.

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.

Effect of Oral Paprika Xanthophyll Intake on Abdominal Fat in Healthy Overweight Humans: A Randomized, Double-blind, Placebo-controlled Study.

PURPOSE: Xanthophylls that exist in various vegetables and fruits have beneficial actions, such as antioxidant activity and an anti-metabolic syndrome effect, and daily intake of xanthophylls could play an important role in preventing lifestyle-related diseases. We investigated whether intake of xanthophylls from red paprika could decrease the abdominal fat area in the healthy overweight volunteers with a body mass index (BMI) ranging from 25 to < 30 kg/m2. METHODS: In a randomized, double-blind, placebo-controlled, parallel-group study, 100 healthy volunteers were assigned to oral administration of paprika xanthophyll capsules (containing 9.0 mg of paprika xanthophylls) or placebo capsules for 12 weeks. The primary endpoint was the effect of paprika xanthophyll intake on the abdominal visceral fat area (VFA) as determined by computed tomography. The secondary endpoints were as follows: 1) changes of the abdominal subcutaneous fat area (SFA), total fat area (TFA), and BMI; 2) changes of lipid metabolism parameters, glucose metabolism parameters, and other blood parameters. RESULTS: After 12 weeks, VFA was smaller in the paprika xanthophyll group than in the placebo group. In the paprika xanthophyll group, there was a significant decrease of SFA, TFA, and BMI after 12 weeks compared with baseline, and the reduction of SFA, TFA, and BMI was significantly greater in the paprika xanthophyll group than in the placebo group. Moreover, total cholesterol and low-density lipoprotein cholesterol decreased significantly in the paprika xanthophyll group, but not in the placebo group. No adverse effects were caused by intake of paprika xanthophyll capsules. CONCLUSIONS: Intake of paprika xanthophylls for 12 weeks significantly reduced the abdominal fat area and BMI in healthy overweight volunteers without causing any adverse effects. These findings suggest that paprika xanthophyll is a safe food ingredient that improves lipid metabolism and reduces abdominal fat. TRIAL REGISTRATION: UMIN-CTR UMIN000021529.

Effects of Dietary Paprika Xanthophylls on Ultraviolet Light-Induced Skin Damage: A Double-Blind Placebo-Controlled Study.

Generation of singlet oxygen by solar ultraviolet (UV) radiation causes acute inflammatory responses in the skin. Accumulation of singlet-oxygen-quenching antioxidants in the skin can suppress this photo-oxidative stress. This study evaluated the effect of dietary xanthophylls from red paprika fruit extract on UV-induced skin damage. A randomised double-blind placebo-controlled parallel group comparison study involving 46 healthy volunteers was performed. The minimal erythema dose (MED) of each individual was determined prior to the study. A capsule containing paprika xanthophylls (9 mg) or a placebo was administered daily for 5 weeks. The MED, minimal tanning dose (MTD), skin physiology parameters (skin color, hydration, and barrier function), and facial skin physiology parameters were evaluated at weeks 0, 2, and 4. The MED of the verum group at 2 and 4 weeks after administration was significantly higher than that of the placebo group. At 4 weeks, the suppression of UV-induced skin darkening by the verum diet was significantly greater than that of the placebo. There were no significant differences in facial skin parameters between the verum and placebo groups. Our results indicate the efficacy of dietary paprika xanthophylls in suppression of UV-induced skin damage.

Accumulation of Paprika Carotenoids in Human Plasma and Erythrocytes.

The accumulation (incorporation) of paprika carotenoid in human plasma and erythrocytes was investigated. A paprika carotenoid supplement (14 mg/day) was ingested for 4 weeks by 5 young healthy volunteers (3 men and 2 women). After 2 weeks of carotenoid ingestion, the carotenoid levels in plasma and erythrocytes increased by 1.2-fold and 2.2-fold, respectively. Characteristic carotenoids found in paprika (capsanthin, cucurbitaxanthin A, and cryptocapsin) were detected in both plasma and erythrocytes. An oxidative metabolite of capsanthin (capsanthone) was also found in both plasma and erythrocytes.

Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L alpha-glucan phosphorylase.

The thermostability of potato type L alpha-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe39-->Leu (F39L), Asn135-->Ser (N135S), and Thr706-->Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60 degrees C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65 degrees C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.

Crystallization and preliminary X-ray crystallographic study of disproportionating enzyme from potato.

Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the alpha-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of alpha-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 A resolution and belongs to space group C222(1), with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 A.