Do Soluble Phosphates Direct the Formose Reaction towards Pentose Sugars?

The formose reaction has been a leading hypothesis for the prebiotic synthesis of sugars such as ribose for many decades but tends to produce complex mixtures of sugars and often tars. Channeling the formose reaction towards the synthesis of biologically useful sugars such as ribose has been a holy grail of origins-of-life research. Here, we tested the hypothesis that a simple, prebiotically plausible phosphorylating agent, acetyl phosphate, could direct the formose reaction towards ribose through phosphorylation of intermediates in a manner resembling gluconeogenesis and the pentose phosphate pathway. We did indeed find that addition of acetyl phosphate to a developing formose reaction stabilized pentoses, including ribose, such that after 5 h of reaction about 10-fold more ribose remained compared with control runs. But mechanistic analyses using liquid chromatography-mass spectrometry showed that, far from being directed towards ribose by phosphorylation, the formose reaction was halted by the precipitation of Ca2+ ions as phosphate minerals such as apatite and hydroxyapatite. Adding orthophosphate had the same effect. Phosphorylated sugars were only detected below the limit of quantification when adding acetyl phosphate. Nonetheless, our findings are not strictly negative. The sensitivity of the formose reaction to geochemically reasonable conditions, combined with the apparent stability of ribose under these conditions, serves as a valuable constraint on possible pathways of sugar synthesis at the origin of life.

Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Identification of dipyrrolone pigments and their precursors formed in the Maillard reaction of carnosine and pentose under weakly acidic conditions.

Colored compounds formed by the Maillard reaction of carnosine with xylose or glucose were investigated in this study. Yellow pigments showing an absorption maximum at 450 nm were found in a heated solution of carnosine with xylose at pH 5.0. These pigments were then isolated and identified as dicarnosyl-dipyrrolones A and B. The generation of dipyrrolones in the absence of lysine suggests that dipyrrolone pigments can be formed by pentose as well as every amino compound such as amino acids, peptides and proteins possessing a free amino group. Analysis of α-dicarbonyls using LC-MS/MS showed that pentosone, 1-deoxypentosone, 3-deoxypentosone (3-DP), and methylglyoxal were predominantly generated via degradation of Amadori compounds. Also, a potential formation pathway of dypyrrolones was established, indicating that an Amadori compound that could form 3-DP is likely to play a role as a main precursor for dipyrrolones.

Crystal structure of a novel homodimeric l-ribulose 3-epimerase from Methylomonus sp.

d-Allulose has potential as a low-calorie sweetener which can suppress fat accumulation. Several enzymes capable of d-allulose production have been isolated, including d-tagatose 3-epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3-epimerase. The synthesized gene encoding the deduced ketose 3-epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward l-ribulose, followed by d-ribulose and d-allulose. The X-ray structure analysis of l-ribulose 3-epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric l-ribulose 3-epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric l-ribulose 3-epimerases, but the short C-terminal α-helix of MetLRE is unique and different from those of known l-ribulose 3 epimerases. The length of the C-terminal α-helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric l-ribulose 3-epimerase identified to exhibit high relative activity toward d-allulose.

Reverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids.

C-Analogues of the canonical N-nucleosides have considerable importance in medicinal chemistry and are promising building blocks of xenobiotic nucleic acids (XNA) in synthetic biology. Although well established for synthesis of N-nucleosides, biocatalytic methods are lacking in C-nucleoside synthetic chemistry. Here, we identify pseudouridine monophosphate C-glycosidase for selective 5-ß-C-glycosylation of uracil and derivatives thereof from pentose 5-phosphate (D-ribose, 2-deoxy-D-ribose, D-arabinose, D-xylose) substrates. Substrate requirements of the enzymatic reaction are consistent with a Mannich-like addition between the pyrimidine nucleobase and the iminium intermediate of enzyme (Lys166) and open-chain pentose 5-phosphate. ß-Elimination of the lysine and stereoselective ring closure give the product. We demonstrate phosphorylation-glycosylation cascade reactions for efficient, one-pot synthesis of C-nucleoside phosphates (yield: 33 - 94%) from unprotected sugar and nucleobase. We show incorporation of the enzymatically synthesized C-nucleotide triphosphates into nucleic acids by RNA polymerase. Collectively, these findings implement biocatalytic methodology for C-nucleotide synthesis which can facilitate XNA engineering for synthetic biology applications.

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides.

In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.

Metabolic engineering for the production of butanol, a potential advanced biofuel, from renewable resources.

Butanol is an important chemical and potential fuel. For more than 100 years, acetone-butanol-ethanol (ABE) fermentation of Clostridium strains has been the most successful process for biological butanol production. In recent years, other microbes have been engineered to produce butanol as well, among which Escherichia coli was the best one. Considering the crude oil price fluctuation, minimizing the cost of butanol production is of highest priority for its industrial application. Therefore, using cheaper feedstocks instead of pure sugars is an important project. In this review, we summarized butanol production from different renewable resources, such as industrial and food waste, lignocellulosic biomass, syngas and other renewable resources. This review will present the current progress in this field and provide insights for further engineering efforts on renewable butanol production.

Identification of a novel recombinant D-lyxose isomerase from Thermoprotei archaeon with high thermostable, weak-acid and nickel ion dependent properties.

Recently, production of D-mannose becomes a hotspot owing to it exhibiting many physiological functions on people's health and wide applications in food and pharmaceutical field. The use of biological enzymes to production of D-mannose is of particular receiving considerable concerns due to it possessing many merits over chemical synthesis and plant extraction strategies. D-Lyxose isomerase (D-LIase) plays a pivotal role in preparation of D-mannose from d-fructose through isomerization reaction. Thus, a novel putative D-LIase from thermophiles strain Thermoprotei archaeon which was expressed in E. coli BL21(DE3) was first identified and biochemically characterized. The recombinant D-LIase showed an optimal temperature of 80 and 85 °C and pH of 6.5. It was highly thermostable at 70 °C and 80 °C after incubating for 48 h and 33 h, respectively, with retaining over 50% of the initial activity. A lower concentration of Ni2+ (0.5 mM) could greatly increase the activity by 25-fold, which was rare reported in other D-LIases. It was a dimer structure with melting temperature of 88.3 °C. Under the optimal conditions, 15.8 g L-1 of D-mannose and 33.8 g L-1 of D-xylulose were produced from 80 g L-1 of d-fructose and D-lyxose, respectively. This work provided a promising candidate sugar isomerase T. archaeon D-LIase for the production of D-mannose and D-xylulose.

Detoxification of sugarcane bagasse hydrolysate with different adsorbents to improve the fermentative process.

Second generation ethanol has the prospect of becoming an important bioenergy alternative. The development of this technology is associated with the lignocellulosic materials' use, with emphasis on agricultural and agroindustrial by-products from which fermentable sugar can be produced. The acid hydrolysis depolymerizes the hemicellulose releasing mainly xylose. Subsequently, the cellulose can be converted into glucose by enzymatic hydrolysis. However, the acid hydrolysis produces toxic compounds, such as furan derivatives, phenolics, and organic acids, which are harmful to fermentative microorganisms. This study investigated different acid concentrations in the sulfuric acid hydrolysis of sugarcane bagasse (1- 5% m/v) and the use of adsorbents with the prerogative to improve the acid hydrolysate (AH) quality for microbial ethanolic fermentation. Cell growth and fermentative yield of Saccharomyces cerevisiae (PE-2) and Scheffersomyces stipitis (NRRL Y-7124) were evaluated. AH was used as a source of pentoses (17.7 g L-1) and molasses (ME) sugarcane as source of hexoses (47 g L-1). The following adsorbents were used: activated charcoal, clay, hydrotalcite and active and inactive cells of PE-2 and NRRL Y-7124, at concentrations ranging (1 - 8% m/v). Results of cell growth and chemical characterization allowed to select the most effective adsorbents with emphasis for active cells that removed 66% furfural and 51% 5-(hydroxymethyl) furfural) (5-HMF) and alcoholic productivity of 23.5 g L-1 in AH and ME substrates, in the presence of mixed culture. These results indicate the application of active yeast cells in the detoxification of acid hydrolysates of the sugarcane bagasse previously to the fermentation.

Characterization of a recombinant l-ribose isomerase from Mycetocola miduiensis and its application for the production of l-ribulose.

An enzyme, l-ribose isomerase (l-RI), mostly catalyzes the isomerization of l-ribose and l-ribulose. These so-called rare sugars are essential for the treatment of cancer and other viral diseases. In the present study, l-ribose isomerase produced from a bacterium, Mycetocola miduiensis (Mm-LRIse), by using l-ribose as a carbon source. The recombinant l-ribose isomerase gene was cloned and overexpressed from M. miduiensis and purified with an exclusive band of 32 kDa by nickel-affinity chromatography. This gene possessed 267 amino acids protein having an estimated molecular weight of 29,568.17 Da. The native molecular weight of Mm-LRIse estimated by HPLC was 134.84 kDa. The recombinant l-ribose isomerase was highly active in sodium phosphate (50 mM) buffer at 40 °C and pH 7.5, showing the specific activity up to 47.40 U mg-1. Mm-LRIse showed no significant enhancement in activity with metallic ions except Mn2+ and Co2+. The values of Km, Kcat, Kcat/Km and Vmax of Mm-LRIse against l-ribose substrate were 42.48 mM, 9259.26 min-1, 217.43 min-1 mM-1, and 277.78 U mg-1 respectively. At equilibrium, the l-ribulose transformation rate was nearly 32 % (6.34 g L-1) using 20 g L-1 of l-ribose. The results revealed that the Mm-LRIse enzyme has a potential for L-ribulose production from l-ribose.

Production and faecal fermentation of pentose oligomers of hemicellulose: Study of variables influencing bioprocess efficiency.

Almond shell, a by-product obtained from the nut industry, was valorised into low degree of polymerisation xylooligosaccharides using alkaline pretreatment and enzymatic hydrolysis. The effect of particle size on hemicellulose recovery upon pretreatment was studied using 1 and 2 M NaOH. It was observed that particle size significantly influences hemicellulose recovery, as particles below 120 µm resulted in near complete recovery at 2 M NaOH. Enzymatic hydrolysis of hemicellulose was optimised using response surface methodology, to obtain efficient xylooligosaccharides production at low enzyme dose and high substrate concentration. For higher XOS yield, an enzyme dose of 10 U and substrate concentration <2% was optimal. The in-vitro human faecal fermentation study revealed no significant difference in gas and short chain fatty acid level among substrates evaluated. It was observed that short chain oligosaccharides produce higher level of acetate than medium chain oligosaccharides.

Six New Methyl Apiofuranosides from the Bark of Phellodendron chinense Schneid and Their Inhibitory Effects on Nitric Oxide Production.

A chemical investigation on 70% EtOH extract from the bark of Phellodendron chinense Schneid (Rutaceae) led to six new methyl apiofuranosides (1-6), and ten known compounds (7-16). All these compounds were characterized by the basic analysis of the spectroscopic data including extensive 1D-, 2D-NMR (HSQC, HMBC), and high-resolution mass spectrometry, and the absolute configurations were determined by both empirical approaches and NOESY. Inhibitory effects of compounds 1-9 and 11-16 on nitric oxide production were investigated in lipopolysaccharide (LPS)-mediated RAW 264.7 cells, as a result, most of these isolates inhibited nitric oxide (NO) release, and among them 9, 11, and 12 displayed the strongest inhibition on NO release at the concentration of 12.5 µM.

Timely Addition of Glutathione for Its Interaction with Deoxypentosone To Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System.

The inhibitory effects of glutathione (GSH) and oxiglutathione (GSSG) on Maillard browning were compared, and it was clarified that free sulfhydryl was the key substance for the inhibition. The Amadori rearrangement product (ARP) derived from glycylglycine (Gly-Gly) and arabinose (Ara) was prepared by aqueous Maillard reaction, and LC-MS/MS was used to investigate the reaction products of GSH and purified ARP. Reaction between GSH and deoxypentosone (DP) was found to alter the pathway of aqueous Maillard reaction, which reduced the production of glyoxal, methylglyoxal, and furfural and thereby inhibited the formation of melanoidins. To determine the optimal conditions for browning inhibition, a stepwise increase of temperature was used to prepare Maillard reaction products (MRPs). The results showed that the optimum browning inhibitory effect was obtained by adding GSH after Gly-Gly and Ara heating at 80 °C for 60 min.

Synthesis of enantiomerically pure enones (2-benzyloxypyran-3-ones) derived from pentoses.

The useful synthons sugar enones (2-benzyloxypyran-3-ones) derived from pentoses have been prepared starting from 2-acetoxyglycals or benzyl pentopyranosides. The glycals were glycosylated with benzyl alcohol in the presence of a Lewis acid (SnCl4 or InCl3) to give enantioenriched enones (ee = 80-90%). Under catalysis with InCl3, benzyl 2-enopyranosides gave also the enones (ee = 87%). On the other hand, enantiomerically pure enones were synthesized via an improved straightforward and high yielding sequence (70% overall) from benzyl pentopyranosides. However, the yields of both, the glycosylation of glycals as well as some specific reactions of the sequence from glycosides, were lowered when a p-nitro substituent was introduced into the benzyl group. These routes became impractical in the case of p-acetamidobenzyl derivatives, because of the large extent of decomposition. Therefore, alternative sequences have been developed for the synthesis of 2-(p-acetamidobenzyloxy)pyran-3-ones.

Alkynylation of Pentose Derivatives with Stereochemical Fidelity: Implications for the Regioselectivity of Alkynyl Diol Cycloisomerizations to Cyclic Enol Ethers.

This work characterizes a previously undetected epimerization in the preparation of alkynyl diols from pentose precursors utilizing the Ohira-Bestmann reagent. Lithium trimethylsilyldiazomethane (Colvin reagent) additions to the d-ribose and d-lyxose-derived benzylidene acetals provide the respective alkynyl diol stereoisomers, without epimerization. Regioselective tungsten-catalyzed cycloisomerizations of the d-ribose- and d-lyxose-derived alkynyl diols yield rigid bicyclic pyranose glycals, confirming the stereochemical fidelity of the Colvin alkynylation process.

Water extractable pentosans - Quantification of ferulic acid using RP-HPLC, techno-rheological and antioxidant properties.

Water extractable pentosans extracted from three varieties of oats were studied for structural analysis using ATR- FTIR, ferulic acid content using RP-HPLC, antioxidant activity by DPPH, reducing power, and metal chelating assays, and functional properties. The appearance of absorption band at 1720 cm-1 in water extractable pentosans is assigned to the presence of aromatic esters as displayed from ATR-FTIR spectrum. All the samples exhibited non-newtonian behavior with viscosities following the order; SWEP > 20WEP > 90WEP. Bile acid binding capacity of water soluble pentosans varied significantly from 46.69 to 49.40%. RP-HPLC displayed that water extractable pentosans from SKO20 contained about 2 times higher FA (423.00 µg/100 g) compared to SWEP (250.00 µg/100 g) and 90 WEP (253.00 µg/100 g). Water soluble pentosans had DPPH scavenging activity, reducing power, and metal chelation activity in the range of 13.57-17.45 (µg α-tocopherol/g), 8.91-10.24 (µg BHT/g), and 0.55-0.76 (µg citric acid/g), respectively.

Phenylalkanoid Glycosides (Non-Salicinoids) from Wood Chips of Salix triandra × dasyclados Hybrid Willow.

Salix triandra (almond leaved willow) is an established crop, grown in coppicing regimes for basket-making materials. It is known as a source of non-salicinoid phenolic glycosides, such as triandrin and salidroside. A spontaneous natural hybrid of S. triandra and S. dasyclados was subjected to metabolite profiling by high resolution LC-MS, and 22 phenolic glycosides, including 18 that are new to the Salicaceae, were identified. Structures were determined by HPLC isolation and NMR methods. The hybridisation process has introduced novel chemistry into the Salix phenolic glycoside palette, in particular, the ability to generate disaccharide conjugates where the glycosyl group is further extended by a range of sugars, including apiose, rhamnose, xylose, and arabinose. Also of note is the appearance of chavicol derivatives, also not previously seen in Salix spp. The work demonstrates the plasticity of the phenolic glycoside biosynthetic pathway, and the potential to improve established crops such as S. triandra and S. dasyclados, via high-value metabolites, for both basketry and bioenergy markets.

One-Pot Bioconversion of l-Arabinose to l-Ribulose in an Enzymatic Cascade.

This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzed by pyranose 2-oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild-type P2O is specific for the oxidation of six-carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l-arabinose at the C2-position. The variant T169G was identified as the best candidate, and this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild-type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4-OH of l-arabinose, which allows a reduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.

X-ray structure of Arthrobacter globiformis M30 ketose 3-epimerase for the production of D-allulose from D-fructose.

The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a D-allulose 3-epimerase (AgD-AE), was determined at 1.96 Šresolution. The crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 103.98, c = 256.53 Å. The structure was solved by molecular replacement using the structure of Mesorhizobium loti L-ribulose 3-epimerase (MlL-RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD-AE formed a homotetramer with twofold symmetry. The overall structure of AgD-AE was more similar to that of MlL-RE than to the known structures of D-psicose (alternative name D-allulose) 3-epimerases (D-PEs or D-AEs), although AgD-AE and MlL-RE have different substrate specificities. Both AgD-AE and MlL-RE have long helices in the C-terminal region that would contribute to the stability of the homotetramer. AgD-AE showed higher enzymatic activity for L-ribulose than D-allulose; however, AgD-AE is stable and is a unique useful enzyme for the production of D-allulose from D-fructose.