Starch biosynthesis: the primer nonreducing-end mechanism versus the nonprimer reducing-end two-site insertion mechanism

Two mechanisms are recognized for polysaccharide chain elongation: (a) the nonreducing-end, primer-dependent mechanism and (b) the reducing-end, two-site insertion mechanism. We recently demonstrated the latter mechanism for starch biosynthesis by pulsing starch granules with ADP-[14C]Glc and chasing with ADPGlc for eight varieties of starch granules. Others have reported the addition of glucose from ADPGlc to the nonreducing ends of maltose, maltotriose, and maltopentaose and a branched maltopentasaccharide. It was concluded that starch chains are biosynthesized by the addition of glucose to the nonreducing ends of maltodextrin primers. In this study, we reinvestigated the maltodextrin reactions by reacting three kinds of starch granules from maize, wheat, and rice with ADP-[14C]Glc in the absence and presence of maltose (G2), maltotriose (G3), and maltodextrin (d.p.12) and found that they inhibited starch biosynthesis rather than stimulating it, as would be expected for primers. The major product in the presence of G2 was G3 with decreasing amounts of G4-G9 and the major products in the presence of G3 was G4 and G5, with decreasing amounts of G6-G9. It was concluded that maltodextrins are acceptors rather than primers. This was confirmed by pulsing the starch granules with ADP-[14C]Glc and chasing with G2, G3, and G6, which gave release of 14C-label from the pulsed granules in the absence of ADPGlc, further demonstrating that maltodextrins are acceptors that inhibit starch biosynthesis by releasing glucose from starch synthase, rather than acting as primers and stimulating biosynthesis.     

Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.     

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. We recently found that the activities of a single alpha-amylase on two different starches were significantly different. We then determined the activities of Bacillus amyloliquefaciens and porcine pancreas alpha-amylases, using eight different starches, solubilized by two methods: autoclaving at 121 degrees C and 1M NaOH at 20 degrees C. There were significant differences in the activities of both of the amylases on all eight of the starches. Previously, it had been found that polyethylene glycol (PEG) stabilized and activated the activities of both enzymes, using a soluble amylose as the substrate. Addition of PEG to the enzymes greatly increased the activities on the eight starches, but the activities still differed significantly. The different activities with the starches were hypothesized as differences in the amounts of secondary and tertiary structures that are partially retained when the different starches are solubilized; the activities on addition of PEG is hypothesized as the formation of highly active species from a series of less active forms.     

Isolation, structure, and characterization of the putative soluble amyloses from potato, wheat, and rice starches

Amylose, a putative linear α-(1→4)-glucan and a component of most starches, was isolated from potato, rice, and wheat starches by forming the 1-butanol complex in a solution of the starches. It previously had been found that these amyloses were incompletely hydrolyzed by β-amylase, indicating that it was partially branched. Solubilization of the butanol complex in water and steam distillation of the 1-butanol, followed by cooling to 4 °C gave precipitation of the double helical, linear, retrograded amylose over a 15 h period, leaving the soluble amylose in solution. The soluble amyloses were precipitated with two volumes of ethanol, and the precipitate was solubilized and reprecipitated to remove traces of linear amylose. The precipitated, soluble amyloses, were partially branched and had properties intermediate between linear amylose and amylopectin. The water solubility of the potato amylose was 10.52 mg/mL, with a number-average degree of polymerization (DP_n) of 8440 and 2.1% branch linkages that had a DP_n of 48; the water solubility of the rice amylose was 8.83 mg/mL, with a DP_n of 2911 and 1.4% branch linkages that had a DP_n of 72; and the water solubility of wheat amylose was 6.33 mg/mL, with a DP_n of 1160 and 1.6% branch linkages that had a DP_n of 64. The three soluble amyloses have structures and properties intermediate between the nearly water insoluble (?1 mg/mL), linear amylose, and the highly water-soluble, 4-5% branched, amylopectin.     

Starch biosynthesis: sucrose as a substrate for the synthesis of a highly branched component found in 12 varieties of starches

D-[14C]glucose was incorporated into starch when 12 varieties of starch granules were incubated with [14C]sucrose. Digestion of the 14C-labeled starches with porcine pancreatic alpha amylase showed that a high percentage (16.1-84.1%) of the synthesized starch gave a relatively high molecular weight alpha-limit dextrin. Hydrolysis of the 12 varieties of starch granules by alpha amylase, without sucrose treatment, also gave an alpha-limit dextrin, ranging in amounts from 0.51% (w/w) for amylomaize-7 starch to 8.47% (w/w) for rice starch. These alpha-limit dextrins had relatively high molecular weights, 2.47 kDa for amylomaize-7 starch to 5.75 kDa for waxy maize starch, and a high degree of alpha-(1-->6) branching, ranging from 15.6% for rice starch to 41.1% for shoti starch. ADPGlc and UDPGlc did not synthesize a significant amount (1-2%) of the branched component, suggesting that sucrose is the probable substrate for the in vivo synthesis of the component and that sucrose is not first converted into a nucleotide-glucose diphosphate intermediate.     

Starch biosynthesis: mechanism for the elongation of starch chains

Starch granules from eight diverse plant sources all had active starch synthases and branching enzymes inside the granules. The enzymes synthesized both amylose and amylopectin from ADPGlc. Pulsing of the granules with ADP-[14C]Glc gave synthesis of starch that on reduction and glucoamylase hydrolysis gave 14C-labeled D-glucitol. The pulsed label could be chased by nonlabeled ADPGlc to give a significant decrease of 14C-label in D-glucitol. Evidence further indicated that the synthase forms a high-energy covalent complex with D-glucose and the growing starch chain, and that the D-glucopyranosyl group is added to the reducing end of the growing starch chain by a two-site insertion mechanism.     

Fijian medicinal plants and their role in the prevention of Type 2 diabetes mellitus

Medicinal plants (MPs) are natural sources of active compounds with potential therapeutic benefits in alleviating various illnesses for decades. Fijian people also are using these MPs for the management/prevention of Type 2 diabetes mellitus (T2DM) and associated complications. However, till date, none of these Fijian MP’s antidiabetic potential have been explored or evaluated. Here, we investigated the antidiabetic potential of Fijian MPs scientifically. Phytochemicals such as polyphenols were detected to inhibit the activity of α-amylase and α-glucosidase, the two key carbohydrate enzymes linked to T2DM. Therefore, in the present study, the total phenolic content (TPC), α-amylase and α-glucosidase inhibitory activity of five Fijian MPs: Vobo (Mussaenda raiateensis, MR), Vula walu (Blechnum orientale, BO), Gasau (Miscanthus floridulus, MF), Molikaro (Citrus limon, CL) and Beki ni sina (Dicranopteris caudate, DC) collected from mainland region of Vitilevu, Fiji Islands, were evaluated in vitro. The hydromethanolic (ME) and dichloromethane (DM) extracts of these selected MPs were investigated. The ME extracts of BO (0.102 ± 0.009 mM CE) and DC (0.098 ± 0.09 mM Catechin Equivalence [CE]) showed a higher TPC compared with the control [vanillic acid (0.052 ± 0.003 mM CE, *P value < 0.05)]. However, the TPC of MF, MR and CL were found in the range of 0.020 ± 0.009 to 0.009 ± 0.01 mM CE. The ME extracts of MF and MR inhibited α-glucosidase significantly in comparison with acarbose as evidenced from the IC₅₀ values (IC₅₀ of MF = 1.58 ± 0.03 ng/µl; IC₅₀ of MR = 1.87 ± 0.43 ng/µl and IC₅₀ of acarbose = 3.34 ± 0.15 ng/µl). Moreover, DM extracts of MR (IC₅₀ = 1.31 ± 0.29 ng/µl) also showed significantly higher α-glucosidase inhibitory activity. In contrary, MR (IC₅₀ = 16.18 ± 0.16 ng/µl) and CL (IC₅₀ = 9.21 ± 0.51 ng/µl) also showed significant α-amylase inhibitory activity in ME and DM extracts, respectively. These, results suggest that Fijian MPs could be a potential source of natural inhibitors of enzymes involved in carbohydrate digestion and thus may possibly be used in managing T2DM.