Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics.

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein-ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC50 was measured (IC50 = 1.738 ± 0.041 mM), and the dissociation constant was determined (Ki Slope = 0.46 ± 0.04 mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. AbbreviationsAKGalpha-ketoglutaric acidpNPG4-nitrophenyl-α-d-glucopyranosideANS1-anilinonaphthalene-8-sulfonateMDmolecular dynamics.Communicated by Ramaswamy H. Sarma.

The inhibitory effect of pyrogallol on tyrosinase activity and structure: Integration study of inhibition kinetics with molecular dynamics simulation.

Pyrogallol is naturally found in aquatic plant and has been proposed as a substrate of tyrosinase. In this study, we evaluated the dual effect of pyrogallol on tyrosinase as an inhibitor in the presence of L­DOPA simultaneously via integrating methods of enzyme kinetics and computational molecular dynamics (MD) simulations. Pyrogallol was found to be a reversible inhibitor of tyrosinase in the presence of L­DOPA and its induced mechanism was the parabolic non-competitive inhibition type (IC50 = 0.772 ±â€¯0.003 mM and Ki = 0.529 ±â€¯0.022 mM). Kinetic measurements by real-time interval assay showed that pyrogallol induced rapid inactivation process composing with slight activations at the low dose. Spectrofluorimetry studies showed that pyrogallol mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational MD simulations further revealed that pyrogallol could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS85, HIS259, ASN260, HIS263, VAL283, and ALA296. Our study provides insight into the mechanism by which hydroxyl group composing pyrogallol inhibit tyrosinase and pyrogallol is a potential natural anti-pigmentation agent.

Inhibitory effect of raspberry ketone on α-glucosidase: Docking simulation integrating inhibition kinetics.

Inhibition of α-glucosidase is directly associated with treatment of type 2 diabetes. In this regard, we conducted enzyme kinetics integrated with computational docking simulation to assess the inhibitory effect of raspberry ketone (RK) on α-glucosidase. RK bound to the active site of α-glucosidase and interacted with several key residues such as ASP68, TYR71, HIS111, PHE157, PHE158, PHE177, GLN181, ASP214, THR215, ASP349, ASP408, and ARG439, as detected by protein-ligand docking simulation. Subsequently, we confirmed the action of RK on α-glucosidase as the non-competitive type of inhibition in a reversible and rapidly binding manner. The relevant kinetic parameters were IC50=6.17±0.46mM and Ki=7.939±0.211mM. Regarding the structure-activity relationship, the higher concentration of RK induced slight modulation of the shape of the active site as monitored by hydrophobic exposure. The tertiary conformational change was linked to RK inhibition, and mostly involved regional changes of the active site. Our study provides insight into the functional role of RK due to its structural property of a hydroxyphenyl ring that interacts with the active site of α-glucosidase. We suggest that similar hydroxyphenyl ring compounds targeting the key residues of the active site might be potential α-glucosidase inhibitors.

Pseudoalteromonas gelatinilytica sp. nov., isolated from surface seawater.

Three Gram-stain-negative, rod-shaped bacteria, designated strains NH153T, F-2-11 and M-1-78, were isolated from surface seawater of the South China Sea and the East China Sea. The three isolates were able to grow at 15-45 °C (optimum 28-37 °C), but no growth occurred at 4 or 50 °C. The pH range for growth was pH 5.5-9.5 (optimum pH 7.5-8.5). The isolates required sea salts for growth and growth occurred in the presence of 0-10 % (w/v) NaCl (optimum 3-5 %); no growth occurred in the presence of 12.0, 15.0 or 20.0 % (w/v) NaCl. They were positive for hydrolysis of gelatin and Tween 80. The sole respiratory quinone was ubiquinone-8 (Q-8). The major cellular fatty acids (>10 %) were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major polar lipid components were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid, one unidentified phospholipid and one unidentified lipid. The genomic DNA G+C content of strain NH153T was 41.4 mol%. Based on 16S rRNA gene sequence analysis, the isolates were closely related to the type strain of Pseudoalteromonas shioyasakiensis (98.0-98.6 % sequence similarity). The 16S rRNA gene sequence similarities between the three isolates were 98.8-99.7 %. Phylogenetic analysis indicated that they formed a distinct lineage and clustered with P. shioyasakiensis and Pseudoalteromonas arabiensis. The level of DNA-DNA relatedness among the three isolates was 78.0-85.5 %. Strain NH153T exhibited average nucleotide identity values of 93.4 and 84.2 % with respect to P. shioyasakiensisJCM 18891T and P. arabiensisJCM 17292T, respectively. The genome-to-genome distance analysis revealed that strain NH153T shared 52.4 % DNA relatedness with P. shioyasakiensisJCM 18891T and 28.1 % with P. arabiensisJCM 17292T. On the basis of the phenotypic, genotypic and chemotaxonomic characterizations, as well as phylogenetic inference obtained in this study, strains NH153T, F-2-11 and M-1-78 represent a novel species within the genus Pseudoalteromonas, for which the name Pseudoalteromonasgelatinilytica sp. nov. is proposed. The type strain is NH153T (=CGMCC 1.15370T=DSM 100951T), and F-2-11 (=CGMCC 1.15364=DSM 100953) and M-1-78 (=CGMCC 1.15365=DSM 100952), are additional strains of the species.

[Studies of on-site night soil and kitchen garbage treatment].

The biological treatment technique of collection at source and disposition on-site of night soil and kitchen garbage were presented. By design project of overall technics, the lab-scale experiments were performed. It was revealed that water consumption of vacuum closestool was about 1 L/time. It consumed 0.4- 0.6L water to shred 1 kg kitchen garbage. Night soil covered 40%, kitchen garbage covered 60% in the influent. Water was controlled at about 93%, the C:N ratio was about 25:1, pH was between 6.2 and 7.3, the optical blend frequency was 6h/d and the overall solid retention time was 28 days in anaerobic digestion reactor. The COD removal rate of mixed supernatant was 91% in anaerobic baffled reactor. It was identified that these phosphorus strains and potassium strains were Bacillus. sp, and biological activated fertilizer was obtained by mixed these strains with digestion sludge which had been dehydrated and deodorized. These strains ability of forming phosphorus and potassium were determined, and the concentration of phosphorus increased 67.5%, potassium increased 33.4%.