X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase.

A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. KEY POINTS: • Crystal structures of LrL-RhI in complexes with substrates were determined. • LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. • The LrL-RhI is active in acidic conditions.

Structural and biochemical characterizations of Thermus thermophilus HB8 transketolase producing a heptulose.

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.

Effect of chronic exposure to ketohexoses on pancreatic ß-cell function in INS-1 rat insulinoma cells.

Glucotoxicity, impaired insulin secretion, suppression of insulin gene expression, and apoptosis, in pancreatic ß-cells caused by chronic hyperglycemia is a key component of the pathogenesis of type 2 diabetes. Recently, it has been reported that rare sugar d-allulose has antihyperglycemic and antihyperlipidemic effects in diabetic rats. However, the direct effects of rare sugars including d-allulose on pancreatic ß-cell function are unclear. In this study, we investigated whether chronic exposure to ketohexoses causes glucotoxicity, suppression of insulin gene expression, and apoptosis, in INS-1 rat pancreatic insulinoma cells. d-Fructose, d-tagatose, l-allulose, and l-sorbose treatment for 1-week reduced insulin gene expression, whereas d-allulose, d-sorbose, l-fructose, and l-tagatose did not. All ketohexoses were transported into INS-1 cells, but were not metabolized. In addition, the ketohexoses did not induce apoptosis and did not affect glucose metabolism. These results suggest that long-term administration of d-allulose, d-sorbose, l-fructose, and l-tagatose does not affect pancreatic ß-cell function.

Production of d-aldotetrose from l-erythrulose using various isomerases.

d-Aldotetroses are rare sugars that are obtained via chemical synthesis in low yield. In this study, we demonstrated that d-aldotetroses could be produced using 3 isomerases. First, l-erythrulose was epimerized using d-tagatose 3-epimerase from Pseudomonas cichorii ST-24. The specific optical rotation of the reaction solution gradually decreased to zero, indicating that approximately 50% of the l-erythrulose was converted to d-erythrulose. d, l-Erythrulose mixture was isomerized with d-arabinose isomerase from Klebsiella pneumoniae 40bXX to produce d-threose, resulting in a conversion rate of 9.35%. d-Erythrose production using l-rhamnose isomerase from Pseudomonas stutzeri LL172 resulted in a conversion rate of 12.9%. Because of the low purity of the purchased d-erythrose, the product was reduced by the Raney nickel catalyst compared with authentic erythritol. We confirmed the products using HPLC and 13C-NMR spectra. This is the first report of d-aldotetrose production using an enzymatic reaction.

Safety evaluation and maximum use level for transient ingestion in humans of allitol.

Allitol is a hexitol produced by reducing the rare sugar D-allulose with a metal catalyst under hydrogen gas. To confirm the safe level of allitol, we conducted a series of safety assessments. From the results of Ames mutagenicity assay using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, Escherichia coli strain WP2uvrA, and an in vitro chromosomal aberration test on cultured Chinese hamster cells, allitol did not show any significant genotoxic effect. No significant effects on general condition, urinalysis, hematology, physiology, histopathology, or at necropsy were observed at a dose of 1500 mg/kg body weight of allitol in the acute and 90-day subchronic oral-toxicity assessments for rats. A further study performed on healthy adult humans showed that the acute use level of allitol for diarrhea was 0.2 g/kg body weight for both men and women. The results of current safety assessment studies suggest that allitol is safe for human consumption.

Antitumor Effects of Orally Administered Rare Sugar D-Allose in Bladder Cancer.

D-allose is a rare sugar that has been reported to up-regulate thioredoxin-interacting protein (TXNIP) expression and affect the production of intracellular reactive oxygen species (ROS). However, the antitumor effect of D-allose is unknown. This study aimed to determine whether orally administered D-allose could be a candidate drug against bladder cancer (BC). To this end, BC cell lines were treated with varying concentrations of D-allose (10, 25, and 50 mM). Cell viability and intracellular ROS levels were assessed using cell viability assay and flow cytometry. TXNIP expression was evaluated using Western blotting. The antitumor effect of orally administered D-allose was assessed using a xenograft mouse model. D-allose reduced cell viability and induced intracellular ROS production in BC cells. Moreover, D-allose stimulated TXNIP expression in a dose-dependent manner. Co-treatment of D-allose and the antioxidant L-glutathione canceled the D-allose-induced reduction in cell viability and intracellular ROS elevation. Furthermore, oral administration of D-allose inhibited tumor growth without adverse effects (p < 0.05). Histopathological findings in tumor tissues showed that D-allose decreased the nuclear fission rate from 4.1 to 1.1% (p = 0.004). Oral administration of D-allose suppressed BC growth in a preclinical mouse model, possibly through up-regulation of TXNIP expression followed by an increase in intracellular ROS. Therefore, D-allose is a potential therapeutic compound for the treatment of BC.

Biochemical synthesis of the medicinal sugar l-gulose using fungal alditol oxidase.

Some rare sugars can be potently medicinal, such as l-gulose. In this study, we present a novel alditol oxidase (fAldOx) from the soil fungus Penicillium sp. KU-1, and its application for the effective production of l-gulose. To the best of our knowledge, this is the first report of a successful direct conversion of d-sorbitol to l-gulose. We further purified it to homogeneity with a ∼108-fold purification and an overall yield of 3.26%, and also determined the effectors of fAldOx. The enzyme possessed broad substrate specificity for alditols such as erythritol (kcat/KM, 355 m-1 s-1), thus implying that the effective production of multiple rare sugars for medicinal applications may be possible.

Efficient production of the rare sugar l-gulose using a wheat-bran culture extract of Penicillium sp. KU-1.

We found that l-gulose, a rare sugar, was produced from d-sorbitol efficiently, using a wheat-bran culture extract of the fungus Penicillium sp. KU-1 isolated from soil. The culture extract showed enzyme activity for the oxidation of d-sorbitol to produce l-gulose; a high production yield of approximately 94% was achieved.

Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay.

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI50) concentration by 1d-d-Alu was estimated to be 5.4 mM, which is approximately 10 times lower than that of d-allulose (52.7 mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5 mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.

d-Allulose, a stereoisomer of d-fructose, extends Caenorhabditis elegans lifespan through a dietary restriction mechanism: A new candidate dietary restriction mimetic.

Dietary restriction (DR) is an effective intervention known to increase lifespan in a wide variety of organisms. DR also delays the onset of aging-associated diseases. DR mimetics, compounds that can mimic the effects of DR, have been intensively explored. d-Allulose (d-Alu), the C3-epimer of d-fructose, is a rare sugar that has various health benefits, including anti-hyperglycemia and anti-obesity effects. Here, we report that d-Alu increased the lifespan of Caenorhabditis elegans both under monoxenic and axenic culture conditions. d-Alu did not further extend the lifespan of the long-lived DR model eat-2 mutant, strongly indicating that the effect is related to DR. However, d-Alu did not reduce the food intake of wild-type C. elegans. To explore the mechanisms of the d-Alu longevity effect, we examined the lifespan of d-Alu-treated mutants deficient for nutrient sensing pathway-related genes daf-16, sir-2.1, aak-2, and skn-1. As a result, d-Alu increased the lifespan of the daf-16, sir-2.1, and skn-1 mutants, but not the aak-2 mutant, indicating that the lifespan extension was dependent on the energy sensor, AMP-activated protein kinase (AMPK). d-Alu also enhanced the mRNA expression and enzyme activities of superoxide dismutase (SOD) and catalase. From these findings, we conclude that d-Alu extends lifespan by increasing oxidative stress resistance through a DR mechanism, making it a candidate DR mimetic.

6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology.

In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

Growth inhibitory effect of D-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide.

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar D-arabinose (D-Ara) showed particularly strong growth inhibition. The IC50 value for D-Ara was estimated to be 7.5 mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-D-glucose (19.5 mM) used as a positive control. The inhibitory effect of D-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of D-Ara. The D-Ara-induced inhibition was recovered by adding either D-ribose or D-fructose, but not D-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of D-ribose and D-fructose metabolism.

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans.

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Enzymatic production of three 6-deoxy-aldohexoses from L-rhamnose.

6-Deoxy-L-glucose, 6-deoxy-L-altrose, and 6-deoxy-L-allose were produced from L-rhamnose with an immobilized enzyme that was partially purified (IE) and an immobilized Escherichia coli recombinant treated with toluene (TT). 6-Deoxy-L-psicose was produced from L-rhamnose by a combination of L-rhamnose isomerase (TT-PsLRhI) and D-tagatose 3-epimerase (TT-PcDTE). The purified 6-deoxy-L-psicose was isomerized to 6-deoxy-L-altrose and 6-deoxy-L-allose with L-arabinose isomerase (TT-EaLAI) and L-ribose isomerase (TT-AcLRI), respectively, and then was epimerized to L-rhamnulose with immobilized D-tagatose 3-epimerase (IE-PcDTE). Following purification, L-rhamnulose was converted to 6-deoxy-L-glucose with D-arabinose isomerase (TT-BpDAI). The equilibrium ratios of 6-deoxy-L-psicose:6-deoxy-L-altrose, 6-deoxy-L-psicose:6-deoxy-L-allose, and L-rhamnulose:6-deoxy-L-glucose were 60:40, 40:60, and 27:73, respectively. The production yields of 6-deoxy-L-glucose, 6-deoxy-L-altrose, and 6-deoxy-L-allose from L-rhamnose were 5.4, 14.6, and 25.1%, respectively. These results indicate that the aldose isomerases used in this study acted on 6-deoxy aldohexoses.

Short and sweet: (D)-glucose to (L)-glucose and (L)-glucuronic acid.

The scarcity and expense of access to L-sugars and other rare sugars have prevented the exploitation of their biological potential; for example D-psicose, only recently available, has been recognized as an important new food. Here we give the definitive and cheap synthesis of 99.4% pure L-glucose from D-glucose which requires purification of neither intermediates nor final product other than extraction into and removal of solvents; a simple crystallization will raise the purity to >99.8%.

Systemically Administered D-allose Inhibits the Tumor Energy Pathway and Exerts Synergistic Effects With Radiation.

BACKGROUND/AIM: The present study investigated the anticancer effects of intraperitoneally administered D-allose in in vivo models of head and neck cancer cell lines. MATERIALS AND METHODS: To assess the direct effects of D-allose, its dynamics in blood and tumor tissues were examined. RESULTS: D-allose was detected in blood and tumor tissues 10 min after its intraperitoneal administration and then gradually decreased. In vivo experiments revealed that radiation plus D-allose was more effective than either treatment alone. Thioredoxin-interacting protein (TXNIP) mRNA over-expression was detected after the addition of D-allose in in vitro and in vivo experiments. D-allose inhibited cell growth, which was associated with decreases in glycolysis and intracellular ATP levels and the prolonged activation of AMPK. The phosphorylation of p38-MAPK was also observed early after the administration of D-allose and was followed by the activation of AMPK and up-regulated expression of TXNIP in both in vitro and in vivo experiments. CONCLUSION: Systemically administered D-allose appears to exert antitumor effects. Further studies are needed to clarify the appropriate dosage and timing of the administration of D-allose and its combination with other metabolic agents.

Structural insight into L-ribulose 3-epimerase from Mesorhizobium loti.

L-Ribulose 3-epimerase (L-RE) from Mesorhizobium loti has been identified as the first ketose 3-epimerase that shows the highest observed activity towards ketopentoses. In the present study, the crystal structure of the enzyme was determined to 2.7 Šresolution. The asymmetric unit contained two homotetramers with the monomer folded into an (α/ß)8-barrel carrying four additional short α-helices. The overall structure of M. loti L-RE showed significant similarity to the structures of ketose 3-epimerases from Pseudomonas cichorii, Agrobacterium tumefaciens and Clostridium cellulolyticum, which use ketohexoses as preferred substrates. However, the size of the C-terminal helix (α8) was much larger in M. loti L-RE than the corresponding helices in the other enzymes. In M. loti L-RE the α8 helix and the following C-terminal tail possessed a unique subunit-subunit interface which promoted the formation of additional intermolecular interactions and strengthened the enzyme stability. Structural comparisons revealed that the relatively small hydrophobic pocket of the enzyme around the substrate was likely to be the main factor responsible for the marked specificity for ketopentoses shown by M. loti L-RE.

Phosphorylation of D-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L.

We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition.