Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 µg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. ABBREVIATIONS: 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

Inhibition of α-amylase and α-glucosidase by Morus australis fruit extract and its components iminosugar, anthocyanin, and glucose.

The inhibition of α-amylase and α-glucosidase are important for the maintenance of blood glucose level. Mammalian α-glucosidase includes maltase-glucoamylase and sucrase-isomaltase complexes. In this study, we examined the inhibitory effects of Morus australis fruit extract and its components, that is, three iminosugars (1-deoxynojirimycin [1-DNJ], fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin), two anthocyanins (cyanidin-3-glucoside and cyanidin-3-rutinoside), and glucose, against α-amylase and α-glucosidase. We also quantified the concentration of each component in M. australis fruit extract. The IC50 values of the fruit extracts of four M. australis subspecies were >10 mg/ml for α-amylase, 1.1-1.7 mg/ml for maltase, 6.9-8.6 mg/ml for glucoamylase, 0.13-1.0 mg/ml for sucrase, and 0.46-1.4 mg/ml for isomaltase. When the IC50 value of each component and the concentration of each component in the fruit extract were taken into consideration, our results indicated that glucose are involved in the inhibition of α-amylase, and 1-DNJ and glucose are involved in the inhibition of α-glucosidase. This is in contrast to that in M. australis leaf, neither anthocyanin nor glucose are contained, and 1-DNJ is a main inhibitor. PRACTICAL APPLICATION: It is widely accepted that inhibition of α-amylase and α-glucosidase is one of the strategies to treat type-2 diabetes. Today, acarbose, miglitol, and voglibose are clinically used for this purpose. Our results that 1-DNJ and anthocyanin are present in Morus australis fruit and are involved in the inhibition of α-amylase and α-glucosidase suggest that M. australis fruit is a healthy sweetener.

Inhibitory effect of Morus australis leaf extract and its component iminosugars on intestinal carbohydrate-digesting enzymes.

α-Amylase and α-glucosidase are central enzymes involved in the digestion of carbohydrates. α-Glucosidase includes maltase-glucoamylase and sucrase-isomaltase. We have previously performed the kinetic analysis of the inhibitory effects of powdered or roasted Morus australis leaf extract and its component iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-d-galactopyranosyl deoxynojirimycin (GAL-DNJ) on the activity of maltase. In this study, we analyzed the inhibitory effects of the aforementioned compounds against α-amylase, glucoamylase, sucrase, and isomaltase. At pH 6.0 and 37 °C, each leaf extract sample inhibited glucoamylase, sucrase, and isomaltase but not α-amylase. 1-DNJ and fagomine showed weak α-amylase inhibitory activity while GAL-DNJ exhibited none. 1-DNJ showed a strong glucoamylase, sucrase, and isomaltase inhibitory potential. The inhibitory potential against these three enzymes was 18-500 and 1500-3000-fold higher in the case of 1-DNJ than that observed in the case of fagomine and GAL-DNJ, respectively. We also observed that the indigestible dextrin could considerably inhibit α-amylase. When the powdered M. australis leaf extract was blended with indigestible dextrin, the mixture inhibited α-amylase, as well as maltase, glucoamylase, sucrase, and isomaltase. These results suggest that the ingestion of the leaf extract blended with indigestible dextrin might have the potential to efficiently suppress the postprandial blood glucose level increase.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae.

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.