Sugar Isomerase Research Articles

Silencing of Glutamine: Fructose-6-Phosphate Aminotransferase Impairs Growth and Development in Sogatella furcifera (Hemiptera: Delphacidae).

Glutamine: fructose-6-phosphate aminotransferase (GFAT), the fourth enzyme in the chitin synthesis pathway, exerts wide-ranging effects on the growth and development of organisms. However, the role of GFAT in Sogatella furcifera remains unknown. In this study, the functional significance of the GFAT gene of S. furcifera was analyzed using a reverse transcription-polymerase chain reaction and RNA interference (RNAi) analyses. The complementary DNA sequence of SfGFAT was 3162 bp in length and contained a 2067 bp open reading frame encoding 688 amino acid residues. Structural domain analysis indicated that the SfGFAT protein consisted of one glutamine aminotransferase class 2 domain and two sugar isomerase domains. Expression profile analysis revealed that SfGFAT was expressed throughout the egg, nymph, and adult phases and was strongly expressed on the first day of each nymph stage and in the integuments of five tissues. RNAi results revealed that SfGFAT gene silencing significantly inhibited the mRNA expression of the target gene and resulted in severe mortality among S. furcifera. In summary, these findings demonstrate that SfGFAT plays a critical role in the development of S. furcifera. Moreover, these results may aid in the development of methods to control the spread of S. furcifera.

Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose-lysine.

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphatestructures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.

Engineering the thermostability of d-lyxose isomerase from Caldanaerobius polysaccharolyticus via multiple computer-aided rational design for efficient synthesis of d-mannose

d-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health. The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market. Some sugar isomerases, such as d-lyxose isomerase (d-LIase), can achieve an isomerization reaction between d-mannose and d-fructose. However, the weak thermostability of d-LIase limits its efficient conversion from d-fructose to d-mannose. Nonetheless, few studies are available that have investigated the molecular modification of d-LIase to improve its thermal stability. In this study, computer-aided tools including FireProt, PROSS, and Consensus Finder were employed to jointly design d-LIase mutants with improved thermostability for the first time. Finally, the obtained five-point mutant M5 (N21G/E78P/V58Y/C119Y/K170P) showed high thermal stability and catalytic activity. The half-life of M5 at 65 °C was 10.22 fold, and the catalytic efficiency towards 600 g/L of d-fructose was 2.6 times to that of the wild type enzyme, respectively. Molecular dynamics simulation and intramolecular forces analysis revealed a thermostability mechanism of highly rigidity conformation, newly formed hydrogen bonds and π-cation interaction between and within protein domains, and redistributed surface electrostatic charges for the mutant M5. This research provided a promising d-LIase mutant for the industrial production of d-mannose from d-fructose.

Development of a sugar isomerase cascade to convert D-xylose to rare sugars

Monosaccharides from biomass can be used as cost-effective raw materials to produce rare sugars which are valuable sweeteners. Enzymes play a pivotal role in such biomass biorefinery processes to develop novel synthetic routes, and improve the environmental friendliness of current routes. Here we propose a proof of concept biorefinery process to prepare useful rare sugars of D-xylulose and D-ribulose from D-xylose which was abundant in biomass. Ribose-5-phosphate isomerase B from Ochrobactrum sp. (OsRpiB) with a unique substrate scope was engineered to intensify the activity of transforming D-xylose to D-xylulose. A double mutant R94N/N137Q with 80% higher catalytic efficiency was identified, and kinetic and molecular dynamic studies were conducted to explore the mechanism underneath. The results revealed a correlation between enhancing stability and activity simultaneously. Then a one-pot enzyme cascade was constructed with OsRpiB and D-tagatose-3-epimerase from Pseudomonas cichorii and transformed D-xylulose further to D-ribulose. The conversions of D-xylose and D-xylulose in cascade reaction were 1.80-fold and 1.83-fold of that in separate reactions with the aid of process parameter adjustment. The presented work is the first reported enzyme cascade converting D-xylose to D-ribulose, and proves the promising role of monosaccharide isomerase platforms in the refinery of biomass.

Membrane-bound D-mannose isomerase of acetic acid bacteria: finding, characterization, and application.

D-Mannose isomerase (EC 5.3.1.7) catalyzing reversible conversion between D-mannose and D-fructose was found in acetic acid bacteria. Cell fractionation confirmed the enzyme to be a typical membrane-bound enzyme, while all sugar isomerases so far reported are cytoplasmic. The optimal enzyme activity was found at pH 5.5, which was clear contrast to the cytoplasmic enzymes having alkaline optimal pH. The enzyme was heat stable and the optimal reaction temperature was observed at around 40 to 60˚C. Purified enzyme after solubilization from membrane fraction showed the total molecular mass of 196 kDa composing of identical four subunits of 48 kDa. Washed cells or immobilized cells were well functional at nearly 80% of conversion ratio from D-mannose to D-fructose and reversely 20-25% of D-fructose to D-mannose. Catalytic properties of the enzyme were discussed with respect to the biotechnological applications to high fructose syrup production from konjac taro.

Elucidating the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine, an Amadori product

Amadori products are stable sugar‐amino acid conjugates that are formed non‐enzymatically via the Maillard reaction that takes place during preparation of foods by heating, roasting, and drying. Fructose‐lysine (F‐Lys, ε‐conjugated) is one of the most abundant Amadori compounds in processed foods and is a key intermediate in the formation of advanced glycation end products, which in turn are implicated in inflammation and disease. The variation among humans in their ability to metabolize F‐Lys has motivated an examination of the inter‐individual differences in gut microbial taxa and the enzymes that help convert F‐Lys into short‐chain fatty acids or cellular energy. Results from such studies are also expected to yield insights into whether F‐Lys utilization by bacterial pathogens (e.g., Escherichia coli, Salmonella enterica serovar Typhimurium) might offer them a competitive edge. Either during or after bacterial uptake, F‐Lys is phosphorylated to form 6‐phosphofructose‐lysine (6‐P‐F‐Lys). FrlB, a deglycase, converts 6‐P‐F‐Lys to L‐lysine and glucose 6‐phosphate, with the latter feeding into glycolysis. Since the catalytic mechanism of FrlB has not been studied, we sought to obtain a high‐resolution structure of Salmonella FrlB ± 6‐P‐F‐Lys and identify the active‐site residues essential for catalysis.After overexpression and purification of recombinant Salmonella FrlB, we obtained its 1.9 Å crystal structure. FrlB exists as a dimer with two identifiable inter‐subunit active sites. In the absence of a co‐crystal structure of FrlB with 6‐P‐F‐Lys, we took two different strategies to delineate its catalytic pocket. First, we observed that the phosphosugar‐binding module–called the sugar isomerase (SIS) domain in FrlB–shared sequence similarity with the eponymous domain in E. coli glucosamine 6‐phosphate synthase (GlmS), which generates glucosamine 6‐phosphate from fructose 6‐phosphate (F‐6‐P) and glutamine. Overlaying the tertiary structures of Salmonella FrlB with E. coli GlmS, which had previously been co‐crystallized with F‐6‐P, helped identify FrlB residues that could be involved in 6‐P‐F‐Lys binding and cleavage. Second, sequence alignment of Salmonella FrlB with FraB, a related and biochemically characterized deglycase required for metabolism of fructose‐asparagine (an Amadori compound) pinpointed residues that could act as a general acid and a general base during deglycation. From these comparative analyses, six candidate residues in FrlB were individually mutated to alanine or another conservative substitution, and the mutant derivatives were purified using affinity chromatography. Our differential scanning fluorimetry studies revealed that all the mutants exhibit thermal stability nearly identical to wild‐type FrlB; importantly, our native mass spectrometry (nMS) studies confirmed that the mutations did not impair the ability of these mutants to form a stable dimer. A spectrophotometric coupled assay was employed to measure the activity of FrlB and the panel of mutants. When a mutation dampened or eliminated deglycase activity, nMS was leveraged to distinguish between a defect in substrate binding versus cleavage. Collectively, our results provide a platform for defining the active site and catalytic mechanism of FrlB even while highlighting the value of exploiting structures of distant homologs to advance structure‐function relationship studies.

Development of a Sugar Isomerase Cascade to Convert D-Xylose to Rare Sugars

Development of a Sugar Isomerase Cascade to Convert D-Xylose to Rare Sugars

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Saccharomyces cerevisiae Requires CFF1 To Produce 4-Hydroxy-5-Methylfuran-3(2H)-One, a Mimic of the Bacterial Quorum-Sensing Autoinducer AI-2.

Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1ΔS. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing.IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.

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.

Insight into natural inhibitors and bridging docking to dynamic simulation against sugar Isomerase (SIS) domain protein.

The pathogen Legionella longbeachae is a causative agent of legionellosis. The antibiotic resistance is the major problem of this modern world. Thus, selective pressure warrants the need for identification of newer drug target. In current study, subtractive proteomics approach screen out SIS (sugar isomerase) domain protein as an attractive receptor molecule for rational drug design. This protein is involved in lipopolysaccharide biosynthesis and catalyzes the isomerization of sedoheptulose 7-phosphate in D-glycero-D-manno-heptose 7-phosphate. Molecular docking revealed compound 1 (2-(6-(N,N-dimethyl sulfamoyl)pipridin-4-yl)pyrazin-2-yl)imidazol-3-ium-1-ide) as the potent inhibitor having GOLD fitness score of 69. The complex is affirmed by half-site effect via simulation analysis. Complex stability was investigated via several approaches that follows dynamic simulation and binding energies. Trajectory analysis revealed slight change in ring positioning of inhibitor inside the active pocket during 130ns (nanosecond). Interestingly, it was affirmed via binding interactions' density distribution. Hence, radial distribution function (RDF) inferred that SER55 and SER83 are the major residues that take part in hydrogen bonding and complex stability. Furthermore, an indigenously developed method axial frequency distribution (AFD) has revealed that ligand moved closer to the active site with both the residues SER55 and SER83 binding to the ligand. The phenomena was observed via rotating motion with respect to receptor center cavity. Thus, inhibitor movement towards allosteric site was observed at the end of simulations. Finally, binding free energy calculations by MMPB/GBSA predicts high compound affinity for the complex. Hence, findings from the current study will aid in the novel drug discovery and future experimental studies. Graphical abstract.

Characterization of an L-Arabinose Isomerase from Bacillus velezensis and Its Application for L-Ribulose and L-Ribose Biosynthesis.

L-Ribulose and L-ribose are two high-value unnatural sugars that can be biosynthesized by sugar isomerases. In this paper, an L-arabinose isomerase (BvAI) from Bacillus velezensis CICC 24777 was cloned and overexpressed in Escherichia coli BL21 (DE3) strain. The maximum activity of recombinant BvAI was observed at 45°C and pH8.0, in the presence of 1.0mM Mn2+. Approximately 207.2g/L L-ribulose was obtained from 300g/L L-arabinose in 1.5h by E. coli harboring BvAI. In addition, approximately 74.25g/L L-ribose was produced from 300g/L L-arabinose in 7h by E. coli co-expressing BvAI and L-RI from Actinotalea fermentans ATCC 43279 (AfRI). This study provides a feasible approach for producing L-ribose from L-arabinose using a co-expression system harboring L-Al and L-RI.

Phosphate sugar isomerases and their potential for rare sugar bioconversion.

Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Characterization of a recombinant D-mannose-producing D-lyxose isomerase from Caldanaerobius polysaccharolyticus

Recently, functional sugars, such as d-mannose, have attracted considerable attention due to their excellent physiological benefits for human health and wide applications in food and pharmaceutical industries. Therefore, d-mannose production using a sugar isomerase such as d-lyxose isomerase (d-LIase) has emerged as a research hotspot owing to its advantages over plant extraction and chemical synthesis methods. In this study, a putative d-LIase gene from Caldanaerobius polysaccharolyticus was cloned and expressed in Escherichia coli. Then, a biochemical characterization of the recombinant d-LIase was carried out and its potential use in d-mannose production also assessed. Results showed that d-LIase exhibited its maximum activity under these optimal conditions: temperature of 65 °C, a pH of 6.5, and the Mn2+ metal ion. The d-LIase was active at pH 6.0–8.0; it was also quite thermostable up to 60 °C and approximately 85 % of its maximum activity was retained after incubating for 4 h. Further, our Nano-DSC analysis determined that its melting temperature (Tm) was 70.74 °C. Using 100, 300, and 500 g L−1 of d-fructose as substrate, 25.6, 74.4, and 115 g L−1 of d-mannose were produced respectively, corresponding to a conversion rate of 25.6 %, 24.8 %, and 23.0 % under optimal conditions. Taken together, our results provide evidence for a promising candidate d-LIase for producing d-mannose directly from d-fructose.

Microbial and enzymatic strategies for the production of L-ribose.

L-Ribose is a non-naturally occurring pentose that recently has become known for its potential application in the pharmaceutical industry, as it is an ideal starting material for use in synthesizing L-nucleosides analogues, an important class of antiviral drugs. In the past few decades, the synthesis of L-ribose has been mainly undertaken through the chemical route. However, chemical synthesis of L-ribose is difficult to achieve on an industrial scale. Therefore, the biotechnological production of L-ribose has gained considerable attention, as it exhibits many merits over the chemical approaches. The present review focuses on various biotechnological strategies for the production of L-ribose through microbial biotransformation and enzymatic catalysis, and in particular on an analysis and comparison of the synthetic methods and different enzymes. The physiological functions and applications of L-ribose are also elucidated. In addition, different sugar isomerases involved in the production of L-ribose from a number of sources are discussed in detail with regard to their biochemical properties. Furthermore, analysis of the separation issues of L-ribose from the reaction solution and different purification methods is presented.Key points • l -Arabinose, l -ribulose and ribitol can be used to produce l -ribose by enzymes. • Five enzymes are systematically introduced for production of l -ribose. • Microbial transformation and enzymatic methods are promising for yielding l -ribose.

Characterization of a d-lyxose isomerase from Bacillus velezensis and its application for the production of d-mannose and l-ribose

d-Mannose and l-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. l-arabinose isomerase catalyzes the conversion of l-arabinose to l-ribulose, while d-lyxose isomerase catalyzes l-ribulose and d-fructose to l-ribose and d-mannose, respectively. In this paper, a putative d-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce d-mannose and l-ribose from d-fructose and l-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co2+. Approximately 110.75 g/L d-mannose was obtained from 500 g/L d-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L l-ribose was obtained from 500 g/L l-arabinose in 8 h by the successive biocatalysis of l-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.

Enhanced production of 2'-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli.

2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has been spotlighted for its neutraceutical and pharmaceutical potentials. Microbial production of 2-FL is promising since it is efficient as compared to other production methods. In 2-FL microbial production via the salvage pathway for biosynthesis of guanosine 5'-diphosphate (GDP)-l-fucose from fucose, the conversion yield from fucose is important because of the high price of fucose. In this study, deletion of the genes (araA and rhaA) coding for arabinose isomerase (AraA) and rhamnose isomerase (RhaA) was attempted in engineered Escherichia coli for improving 2-FL production by using fucose, lactose, and glycerol. The engineered E. coli constructed previously is able to express fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori and deficient in β-galactosidase (LacZ), fucose isomerase (FucI), and fuculose kinase (FucK). The additional double-deletion of the araA and rhaA genes in the engineered E. coli enhanced the product yield of 2-FL to 0.52 mole 2-FL/mole fucose, and hence the concentration of 2-FL reached to 47.0 g/L, which are 44% and two-fold higher than those (23.1 g/L and 0.36 mole 2-FL/mole fucose) of the control strain in fed-batch fermentation. Elimination of sugar isomerases exhibiting promiscuous activities with fucose might be critical in the microbial production of 2-FL through the salvage pathway of GDP-l-fucose.

A Genetic Model to Study Increased Hexosamine Biosynthetic Flux.

Recently, we identified harvest moon (hmn), a fully penetrant and expressive recessive zebrafish mutant with hepatic steatosis. Larvae showed increased triacylglycerol in the absence of other obvious defects. When we attempted to raise these otherwise normal-appearing mutants to adulthood, we observed a developmental arrest and death in the early juvenile period. In this study, we report the positional cloning of the hmn locus and characterization of the defects caused by the mutation. Using bulk segregant analysis and fine mapping, we find that hmn mutants harbor a point mutation in an invariant residue within the sugar isomerase 1 domain of the gene encoding the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP) glutamine-fructose-6-phosphate transamidase (Gfpt1). The mutated protein shows increased abundance. The HBP generates β-N-acetyl-glucosamine (GlcNAc) as a spillover pathway from glucose. GlcNAc can be O-linked to seryl and threonyl residues of diverse cellular proteins (O-GlcNAc modification). Although some of these O-GlcNAc modifications serve an essential structural role, many others are dynamically generated on signaling molecules, including several impacting insulin signaling. We find that gfpt1 mutants show global increase in O-GlcNAc modification, and, surprisingly, lower fasting blood glucose in males. Taken together with our previously reported work, the gfpt1 mutant we isolated demonstrates that global increase in O-GlcNAc modification causes some severe insulin resistance phenotypes (hepatic steatosis and runting) but does not cause hyperglycemia. This animal model will provide a platform for dissecting how O-GlcNAc modification alters insulin responsiveness in multiple tissues.

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity.IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the characterization and functional annotation of a putative nonphosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent, bifunctional, sugar-isomerizing activity. This hyperthermophilic R3E exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from those of its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70°C. Therefore, TM0416 can be functionally classified as a novel type of promiscuous R3E with a potential for the production of rare sugars for the food and pharmaceutical industries.

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.