The molecular basis of sugar detection by an insect taste receptor.

Animals crave sugars because of their energy potential and the pleasurable sensation of tasting sweetness. Yet all sugars are not metabolically equivalent, requiring mechanisms to detect and differentiate between chemically similar sweet substances. Insects use a family of ionotropic gustatory receptors to discriminate sugars1, each of which is selectively activated by specific sweet molecules2-6. Here, to gain insight into the molecular basis of sugar selectivity, we determined structures of Gr9, a gustatory receptor from the silkworm Bombyx mori (BmGr9), in the absence and presence of its sole activating ligand, D-fructose. These structures, along with structure-guided mutagenesis and functional assays, illustrate how D-fructose is enveloped by a ligand-binding pocket that precisely matches the overall shape and pattern of chemical groups in D-fructose. However, our computational docking and experimental binding assays revealed that other sugars also bind BmGr9, yet they are unable to activate the receptor. We determined the structure of BmGr9 in complex with one such non-activating sugar, L-sorbose. Although both sugars bind a similar position, only D-fructose is capable of engaging a bridge of two conserved aromatic residues that connects the pocket to the pore helix, inducing a conformational change that allows the ion-conducting pore to open. Thus, chemical specificity does not depend solely on the selectivity of the ligand-binding pocket, but it is an emergent property arising from a combination of receptor-ligand interactions and allosteric coupling. Our results support a model whereby coarse receptor tuning is derived from the size and chemical characteristics of the pocket, whereas fine-tuning of receptor activation is achieved through the selective engagement of an allosteric pathway that regulates ion conduction.

An artificial multienzyme cascade for the whole-cell synthesis of rare ketoses from glycerol.

PURPOSE: In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. METHODS: To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. RESULTS: The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l-1 only from the sole carbon source glycerol. CONCLUSION: This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.

NADH oxidase from Lactobacillus reuteri: A versatile enzyme for oxidized cofactor regeneration.

Pyridine nucleotide cofactors play important roles in biocatalytic processes that generate value-added chemicals for the pharmaceutical and food industries. Because of the high price of these pyridine cofactors, cofactor regeneration is highly desirable. However, recycling the oxidized form of cofactors, especially NADP+, remains a challenge. Here, we cloned and characterized an NADH oxidase from Lactobacillus reuteri (LreNox) which can oxidize both NADH and NADPH. Unlike many other Noxs, LreNox showed equal catalytic efficiency towards NADH and NADPH. To the best our knowledge, LreNox has the highest activity towards NADPH as a substrate compared to other wild type Noxs. Homology modeling and substrate docking studies provided insights into the dual substrate specificity of LreNox. Gly155, Ser179, and His184 in the LreNox substrate binding pocket, which are absent in other Noxs structures, are crucial for NADPH recognition, providing more space for interactions with the additional phosphate group present in NADPH. We also explored the utility of LreNox for NADP+ regeneration in l-sorbose production by coupling it with a sorbitol dehydrogenase. The turn over number (TTN) improved ~53-fold after using LreNox as the NADP+ recycling enzyme. This study demonstrates that LreNox could potentially be used for the regeneration of NAD(P)+ in commercial applications.

Sweet Structural Signatures Unveiled in Ketohexoses.

The conformational behaviour of naturally occurring ketohexoses has been revealed in a supersonic expansion by Fourier transform microwave spectroscopy coupled with a laser ablation source. Three, two and one conformers of d-tagatose, d-psicose and l-sorbose, respectively, have been identified by their rotational constants extracted from the analysis of the spectra. Singular structural signatures involving the hydroxyl groups OH(1) and OH(2) have been disentangled from the intricate intramolecular hydrogen bond networks stabilising the most abundant conformers. The present results place the old Shallenberger and Kier sweetness theories on a firmer footing.

A two-step strategy for the preparation of 6-deoxy-l-sorbose.

A two-step enzymatic strategy for the efficient and convenient synthesis of 6-deoxy-l-sorbose was reported herein. In the first reaction step, the isomerization of l-fucose (6-deoxy-l-galactose) to l-fuculose (6-deoxy-l-tagatose) catalyzed by l-fucose isomerase (FucI), and the epimerization of l-fuculose to 6-deoxy-l-sorbose catalyzed by d-tagatose 3-epimerase (DTE) were coupled with the targeted phosphorylation of 6-deoxy-l-sorbose by fructose kinase from human (HK) in a one-pot reaction. The resultant 6-deoxy-l-sorbose 1-phosphate was purified by silver nitrate precipitation method. In the second reaction step, the phosphate group of the 6-deoxy-l-sorbose 1-phosphate was hydrolyzed with acid phosphatase (AphA) to produce 6-deoxy-l-sorbose in 81% yield with regard to l-fucose.

Synthesis of an Orthogonally Protected Polyhydroxylated Cyclopentene from l-Sorbose.

The use of l-sorbose in the synthesis of functionalized cyclopentene derivatives was accomplished. These cyclopentene derivatives are related to those found in naturally occurring jatrophane frameworks and in other bioactive compounds. The formation of allyl α-l-sorbopyranoside was a key synthetic step. Regioselective introduction of protecting groups was followed by the hydrolysis of the allyl glycoside to furnish a fully protected acyclic l-sorbose derivative. This acyclic intermediate was subsequently used to give an orthogonally protected polyhydroxylated cyclopentene, which has potential for further synthesis of bioactive compounds. The protected cyclopentene itself showed a clear cytotoxic activity when tested against a panel of human cancer cell lines (HT29, LS174T, SW620, A549, and HeLa cells).

AuPt Alloy on TiO2: A Selective and Durable Catalyst for L-Sorbose Oxidation to 2-Keto-Gulonic Acid.

Pt nanoparticles were prepared by a sol immobilization route, deposited on supports with different acid/base properties (MgO, activated carbon, TiO2 , Al2O3, H-Mordenite), and tested in the selective oxidation of sorbose to 2-keto-gulonic acid (2-KGUA), an important precursor for vitamin C. In general, as the basicity of the support increased, a higher catalytic activity occurred. However, in most cases, a strong deactivation was observed. The best selectivity to 2-KGUA was observed with acidic supports (TiO2 and H-Mordenite) that were able to minimize the formation of C1/C2 products. We also demonstrated that, by alloying Pt to Au, it is possible to enhance significantly the selectivity of Pt-based catalysts. Moreover, the AuPt catalyst, unlike monometallic Pt, showed good stability in recycling because of the prevention of metal leaching during the reaction.

Enzymatic synthesis of rare sugars with L-rhamnulose-1-phosphate aldolase from Thermotoga maritima MSB8.

L-Rhamnulose-1-phosphate aldolase from a thermophilic source (Thermotoga maritima MSB8) (RhaDT.mari) was heterologously overexpressed in Escherichia coli and the stereoselectivity of this enzyme with or without Nus tag was investigated. We also applied this enzyme to the synthesis of rare sugars D-psicose, D-sorbose, L-tagatose and L-fructose using our one-pot four-enzyme system. To the best of our knowledge, this is the first use of RhaD from a thermophilic source for rare sugar synthesis and the temperature tolerance of this enzyme paves the path for large scale fermentation.

Selective electrocatalytic oxidation of sorbitol to fructose and sorbose.

A new electrocatalytic method for the selective electrochemical oxidation of sorbitol to fructose and sorbose is demonstrated by using a platinum electrode promoted by p-block metal atoms. By the studying a range of C4, C5 and C6 polyols, it is found that the promoter interferes with the stereochemistry of the polyol and thereby modifies its reactivity.

Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to 2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.

Ketogulonicigenium vulgare WSH-001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)-dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ-dependent L-sorbose dehydrogenases (SDH) or L-sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ-dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L-sorbose or L-sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ-dependent dehydrogenases expanded the PQQ-dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one-step production of vitamin C in the future.

A reinvestigation of the synthesis and revision of spectral data of 1,2-O-isopropylidene-α-l-sorbofuranose, 1,2:4,6-di-O-isopropylidene-α-L-sorbofuranose and derivatives.

Mono- and di-O-isopropylidene-l-sorbofuranose derivatives are important starting materials for the synthesis of modified sugars and useful chiral compounds. However, several inconsistencies in the spectral data of these compounds and erroneous structural assignments have been noted in the literature. The unambiguous synthesis of 1,2:4,6-di-O-isopropylidene-α-L-sorbofuranose and derivatives of 1,2- and 2,3-O-isopropylidene-α-L-sorbofuranoses has been achieved and definitive spectral data on these compounds are provided.

Stereoselective synthesis of (2S,3S,4R,5S)-3,4-dihydroxy-2,5-dihydroxymethyl pyrrolidine from L-sorbose.

One of the most frequently synthesized iminosugar derivatives is DMDP. Starting from L-sorbose, a practical method for the synthesis of derivatives of this five-membered iminocyclitol has been developed, involving straightforward steps and a convenient selective reduction of a ketoxime intermediate.

Mechanism of mutarotation in supercooled liquid phase: Studies on L-sorbose.

We have studied mutarotation in anhydrous supercooled L-sorbose by means of dielectric spectroscopy. The phenomenon observed in L-sorbose is much faster than in the structurally similar D-fructose. The kinetics of this process has been determined by applying 1st order kinetics model. Activation energy equal to 68 kJ/mol was obtained from temperature dependence of rate constants. To understand differences in mutarotation rate between D-fructose and L-sorbose, quantum mechanical calculations were performed to study mechanism of this phenomenon. The possible impact of water absorbed from air on the mutarotation in supercooled liquid state has been checked. It turned out that the process is probably intermolecular and the water molecules or other carbohydrate molecules assist in the proton transfer process. Finally we have shown that the rate constant can be alternatively determined from frequency of the maximum of peak, obtained by performing Fourier transform of kinetic curve.

Enzymatic synthesis of D-sorbose and D-psicose with aldolase RhaD: effect of acceptor configuration on enzyme stereoselectivity.

It was previously reported that DHAP-dependent aldolase RhaD selectively chooses L-glyceraldehyde from racemic glyceraldehyde to produce l-fructose exclusively. Contrastingly, we discovered that D-glyceraldehyde is also tolerated as an acceptor and the stereoselectivity of the enzyme is lost in the corresponding aldol addition. Furthermore, we applied this property to efficiently synthesize two rare sugars D-sorbose and D-psicose.

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose.

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.

A rare sugar, d-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa.

We investigated responses of rice plant to three rare sugars, d-altrose, d-sorbose, and d-allose, due to establishment of mass production methods for these rare sugars. Root growth and shoot growth were significantly inhibited by d-allose but not by the other rare sugars. A large-scale gene expression analysis using a rice microarray revealed that d-allose treatment causes a high upregulation of many defense-related, pathogenesis-related (PR) protein genes in rice. The PR protein genes were not upregulated by other rare sugars. Furthermore, d-allose treatment of rice plants conferred limited resistance of the rice against the pathogen Xanthomonas oryzae pv. oryzae but the other tested sugars did not. These results indicate that d-allose has a growth inhibitory effect but might prove to be a candidate elicitor for reducing disease development in rice.

Heteroaromatic N-base ligands in 1,10-phenanthroline- and 2,2'-bipyridyl-assisted chromic acid oxidation of (-)-L-sorbose in aqueous micellar acid media: a kinetic study.

In aqueous sulfuric acid media, Cr(VI) oxidation of (-)-L-sorbose in the presence and absence of catalysts like 1,10-phenanthroline (phen), 2,2'-bipyridyl (bipy) have been carried out under the conditions, [(-)-L-sorbose](T)>>[Cr(VI)](T) at different temperatures. Under the experimental conditions, the monomeric species of Cr(VI) has been found kinetically active in the absence of phen and bipy catalysts, while in the heteroaromatic N-base catalysed path, the Cr(VI)-phen and Cr(VI)-bipy complexes have been suggested as the active oxidants. In the catalysed path, Cr(VI)-L complex (L=phen, bipy) receives a nucleophilic attack by the substrate to form a ternary complex which subsequently experiences a redox decomposition through two-electron transfer leading to the organic products and a Cr(IV)-L complex. Both the uncatalysed and catalysed paths show first-order dependence on [(-)-L-sorbose](T) and [Cr(VI)](T). The uncatalysed path shows second-order in [H(+)], while the catalysed path shows a first-order dependence on [H(+)]. The heteroaromatic N-base catalysed path is first-order in [phen](T) or [bipy](T). These findings remain unchanged in the presence of externally added surfactants. The cationic surfactant (i.e., N-cetylpyridinium chloride (CPC)) inhibits the rate in both the catalysed and uncatalysed paths, whereas the anionic surfactant (i.e., sodium dodecyl sulfate (SDS)) shows the rate accelerating effect for both the uncatalysed and catalysed paths. The observed micellar effects have been rationalised by considering the distribution of the reactants between the micellar and aqueous phases in terms of the proposed reaction mechanism.

Studies on the synthesis of valienamine and 1-epi-valienamine starting from D-glucose or L-sorbose.

Two synthetic routes to a carbocyclic precursor to valienamine are reported, starting from either D-glucose or L-sorbose and using ring-closing metathesis as a key step. A low-yielding synthesis of 1-epi-valienamine is reported. Results from an abortive third possible route to valienamine based on an early introduction of nitrogen are discussed.

Potential anthelmintic: D-psicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans.

No anthelmintic sugars have yet been identified. Eight ketohexose stereoisomers (D- and L-forms of psicose, fructose, tagatose and sorbose), along with D-galactose and D-glucose, were examined for potency against L1 stage Caenorhabditis elegans fed Escherichia coli. Of the sugars, D-psicose specifically inhibited the motility, growth and reproductive maturity of the L1 stage. D-Psicose probably interferes with the nematode nutrition. The present results suggest that D-psicose, one of the rare sugars, is a potential anthelmintic.

The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group.

A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of Guillain-Barré syndrome, had unidentified labile, capsular polysaccharide (CPS) structures. In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups. Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-beta-D-GlcA6NGro-(1-3)-beta-D-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol. A labile alpha-sorbofuranose branch located at C2 of GlcA was determined to have the L configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan. A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group. Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain. These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes. The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C. jejuni.