Maltopentaose

Inhibition of Arabidopsis chloroplast beta-amylase BAM3 by maltotriose suggests a mechanism for the control of transitory leaf starch mobilisation.[Pubmed:28225829]

PLoS One. 2017 Feb 22;12(2):e0172504.

Starch breakdown in leaves at night is tightly matched to the duration of the dark period, but the mechanism by which this regulation is achieved is unknown. In Arabidopsis chloroplasts, beta-amylase BAM3 hydrolyses transitory starch, producing maltose and residual maltotriose. The aim of the current research was to investigate the regulatory and kinetic properties of BAM3. The BAM3 protein was expressed in Escherichia coli and first assayed using a model substrate. Enzyme activity was stimulated by treatment with dithiothreitol and was increased 40% by 2-10 muM Ca2+ but did not require Mg2+. In order to investigate substrate specificity and possible regulatory effects of glucans, we developed a GC-MS method to assay reaction products. BAM3 readily hydrolysed maltohexaose with a Km of 1.7 mM and Kcat of 4300 s-1 but activity was 3.4-fold lower with Maltopentaose and was negligible with maltotetraose. With maltohexaose or amylopectin as substrates and using [UL-13C12]maltose in an isotopic dilution method, we discovered that BAM3 activity is inhibited by maltotriose at physiological (mM) concentrations, but not by maltose. In contrast, the extracellular beta-amylase of barley is only weakly inhibited by maltotriose. Our results may explain the impaired starch breakdown in maltotriose-accumulating mutants such as dpe1 which lacks the chloroplast disproportionating enzyme (DPE1) metabolising maltotriose to glucose. We hypothesise that the rate of starch breakdown in leaves can be regulated by inhibition of BAM3 by maltotriose, the concentration of which is determined by DPE, which is in turn influenced by the stromal concentration of glucose. Since the plastid glucose transporter pGlcT catalyses facilitated diffusion between stroma and cytosol, changes in consumption of glucose in the cytosol are expected to lead to concomitant changes in plastid glucose and maltotriose, and hence compensatory changes in BAM3 activity.

Paenibacillus sp. 598K 6-alpha-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from alpha-(1 --> 4)-glucan.[Pubmed:28224195]

Appl Microbiol Biotechnol. 2017 May;101(10):4115-4128.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an alpha-(1 --> 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending alpha-(1 --> 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: alpha-(1 --> 4)-> alpha-(1 --> 2)- > alpha-(1 --> 3)- > alpha-(1 --> 6)-glucobiose and Maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by (13)C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong alpha-(1 --> 4) to alpha-(1 --> 6) transglucosylation activity. Ps6GT31A elongated alpha-(1 --> 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize alpha-(1 --> 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

Self-Assembled Polypeptide Nanogels with Enzymatically Transformable Surface as a Small Interfering RNA Delivery Platform.[Pubmed:29059529]

Biomacromolecules. 2017 Dec 11;18(12):3913-3923.

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing Maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral zeta potential values that became positive after degradation by alpha-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after alpha-amylase exposure. The nanogels, with or without alpha-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With alpha-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities.[Pubmed:28455338]

Appl Environ Microbiol. 2017 Jun 16;83(13). pii: AEM.00038-17.

Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by alpha-1,4- but also some alpha-1,6-linked glucosyl residues. The alpha-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6''-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of alpha-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered alpha-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic alpha-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing alpha-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one alpha-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of Maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various alpha-1,4- and alpha-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing alpha-glucosidase with relaxed linkage specificity (alpha-1,4 and alpha-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.

Does high pressure have any effect on the structure of alpha amylase and its ability to binding to the oligosaccharides having 3-7 residues? Molecular dynamics study.[Pubmed:29328994]

J Mol Graph Model. 2018 Mar;80:85-94.

Studies have shown that deletion of amino acids from the C-terminus of amylase do not alter its amylolytic activity. Although high pressure is used to modify the structure and function of this enzyme, the effects of high pressures on the structures of the wild-type and truncated amylases have not yet been understood at the molecular level. Using molecular dynamic simulations and docking, we studied the structures of wild-type and truncated Taka-amylases at high pressures (1000-4000bar). To construct the truncated Taka-amylase, 50 and 100 C-terminal residues were removed in two separate steps. Results of simulation showed that, although the overall shape partly agglomerates with rise in pressure, high pressure fails to modify the structure of the barrel-like region of the beta-sheet in the wild-type and truncated enzymes. A comparison of contact graphs revealed that the changes at the N-terminus were less extensive than those at the C-terminus. Further analysis showed that 10 regions of the secondary structures changed due to pressure change in wild-type amylase, of which 6 regions were associated with the loops and 4 with helix, while the structure of beta-sheets remained unchanged. The docking of maltotriose, maltotetraose, Maltopentaose, maltohexaose, and maltoheptaose with the averaged structures obtained from different simulations was conducted to characterize the influence of pressure on the activities of the wild-type and truncated enzymes. The results showed that maltoheptaose made hydrophobic contacts with residues Tyr238-Asp117-Tyr82-Leu166-Leu232-Tyr155 and hydrogen contacts with residues Asp233-Gly234-Asp206-Arg204-His296-Glu230. Similar results were obtained for other malto-oligosaccharides.

Study on the Structural Effect of Maltoligosaccharides on Cytochrome c Complexes Stabilities by Native Mass Spectrometry.[Pubmed:29380206]

Nat Prod Bioprospect. 2018 Feb;8(1):57-61.

Noncovalent interactions between ligands and targeting proteins are essential for understanding molecular mechanisms of proteins. In this work, we investigated the interaction of Cytochrome c (Cyt c) with maltoligosaccharides, namely maltose (Mal II), maltotriose (Mal III), maltotetraose (Mal IV), Maltopentaose (Mal V), maltohexaose (Mal VI) and maltoheptaose (Mal VII). Using electrospray ionization mass spetrometry (ESI-MS) assay, the 1:1 and 1:2 complexes formed by Cyt c with maltoligosaccharide ligand were observed. The corresponding association constants were calculated according to the deconvoluted spectra. The order of the relative binding affinities of the selected oligosaccharides with Cyt c were as Mal III > Mal IV > Mal II > Mal V > Mal VI > Mal VII. The results indicated that the stability of noncovalent protein complexes was intimately correlated to the molecular structure of bound ligand. The relevant functional groups that could form H-bonds, electrostatic or hydrophobic forces with protein's amino residues played an important role for the stability of protein complexes. In addition, the steric structure of ligand was also critical for an appropriate interaction with the binding pocket of proteins.

Maltopentaose is the shortest chain oligosaccharide that can be classified as maltodextrin and is also used in a study to investigate glycation and phosphorylation of α-lactalbumin.

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