Mouse AKR1E1 Is an Ortholog of Pig Liver NADPH Dependent 1,5-Anhydro-D-Fructose Reductase

In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.     

Synthesis of 4-O-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-O-isopropylidene-d-fructose

Four novel disaccharides of glycosylated 1,5-anhydro-d-ketoses have been prepared: 1,5-anhydro-4-O-β-d-glucopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-galactopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-glucopyranosyl-d-tagatose, and 1,5-anhydro-4-O-β-d-galactopyranosyl-d-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-β-d-fructopyranose, was prepared from d-fructose and was converted into the d-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.     

1,5-anhydro-D-fructose from D-fructose

1,5-Anhydro-d-fructose was efficiently prepared from d-fructose via regiospecific 1,5-anhydro ring formation of 2,3-O-isopropylidene-1-O-methyl(tolyl)sulfonyl-d-fructopyranose and subsequent deprotection.     

1,5-Anhydro-d-fructose: biocatalytic and chemical synthetic methods for the preparation, transformation and derivatization

1,5-Anhydro-d-fructose (1,5AnFru) is a monoketosaccharide that can be prepared enzymatically from starch by α-1,4-glucan lyase or chemically from d-glucose or d-fructose in a few steps with high yields. The formed 1,5AnFru can be derivatized both enzymatically and chemically to interesting new carbohydrate derivatives, some with biological activities. For example dehydratases, isomerases and reductases can convert 1,5AnFru to enolones (as Ascopyrone P) and sugar alcohols with antimicrobial and antioxidant properties, while chemical modifications can give similar compounds as well as natural products like 1-deoxymannonojirimycin and Clavulazine. 1,5AnFru disaccharides (glycosyl 1→4 1,5AnFru) have been prepared as well as glycosyl 1→4 1,5-anhydro-d-tagatose.     

Action of Nitrile Hydratase from Rhodococcus rhodochrous IFO 15564 on Derivatives of 2,5-Anhydro-d-allononitrile

The conversion of 2,5-anhydro-d-allononitrile derivatives by a nitrile hydratase from Rhodococcus rhodochrous IFO 15564 was studied. The activity of the enzyme was strongly effected by the steric bulkiness of the substituents at the 3-position of the substrates, and the corresponding amides were obtained in high yields from the nitriles with free hydroxyl groups at the 3- and 4-positions.     

A stereocontrolled synthesis of 3-acetamido-1,3,5-trideoxy- and 1,3,5,6-tetradeoxy-1,5-imino-d-glucitol

3-Acetamido-5-amino-3,5,6-trideoxy-d-glucono-1,5-lactam and 3-acetamido-5-amino-3,5-dideoxy-d-glucono-1,5-lactam were synthesized from corresponding 3-acetamido-3-deoxy-β-d-glucopyranosides in 63% and 35% overall yield, respectively. Acetylation followed by reduction led to the title 3-acetamido-3-deoxy derivatives of both deoxynojirimycin and 1,6-dideoxynojirimycin. The procedure developed is useful for a multi-gram scale.     

A highly specific glyoxylate reductase derived from a formate dehydrogenase

A Glu141Asn mutant Paracoccus sp. 12-A formate dehydrogenase catalyzes marked glyoxylate reduction. Additional replacement of the His332-Gln313 pair with His-Glu, which is a consensus acid/base catalyst in D-hydroxyacid dehydrogenases, further improved the catalytic activity of the enzyme as to glyoxylate reduction through enhancement of the hydrogen transfer step in the catalytic process, slightly shifting the optimal pH for the reaction. On the other hand, the replacement induced no marked activity toward other 2-ketoacid substrates, and diminished the enzyme activity as to formate oxidation. Consequently, the formate dehydrogenase was converted to a highly specific and active glyoxylate reductase through only the two amino acid replacements.     

Purification and Properties of Two Chitinases from Streptomyces sp. J-13-3

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.     

Production of 1,5-anhydro-d-fructose by an α-glucosidase belonging to glycoside hydrolase family 31

α-1,4-Glucan lyases [glycoside hydrolase family (GH) 31] catalyze an elimination reaction to form 1,5-anhydro-d-fructose (AF), while GH31 α-glucosidases normally catalyze a hydrolytic reaction. We determined that a small amount of AF was produced by GH31 Aspergillus niger α-glucosidase from maltooligosaccharides by elimination reaction, likely via an oxocarbenium ion intermediate.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F

1,5-Anhydro-d-fructose (AF), a metabolite of starch/glycogen degradation, is a good antioxidant. With the prospect of increasing its applications and use as a food ingredient, AF glucosylation catalysed by the dextransucrase from Leuconostoc mesenteroides NRRL B-512F was performed in the presence of sucrose. This led to AF glucosylated derivatives containing alpha-(1-->6) linkages named 1,5-anhydro-d-fructo-glucooligosaccharides (AFGOS). LC-MS analyses showed that AFGOS with a degree of polymerisation (DP) of up to 7 were synthesised. The amount of AFGOS produced and the average DP increased by using a high sucrose/AF molar ratio and high total sugar concentration. AFGOS were proved to present antioxidant properties quite similar to AF.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

A new chemical synthesis of Ascopyrone P from 1,5-anhydro-D-fructose

The naturally occurring antioxidant Ascopyrone P (1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, 1) was prepared from the rare sugar 1,5-anhydro-D-fructose (AF, 3) in three steps in an overall yield of 36%. Thus, acetylation of 3 afforded the enolone 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulopyranose (4), which could be isomerised to 2,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-1-ene-3-ulose (6). Deacetylation of 6 under mild conditions gave crystalline Ascopyrone P (1).     

Confirmation of the structure of tetra-O-(tert-butyldimethylsilyl)-D-glucono-1,4-lactone formed by silylation of D-glucono-1,5-lactone

The structure of tetra-O-(tert-butyldimethylsilyl)-D-glucono-1,4-lactone made by the silylation of D-glucono-1,5-lactone has been confirmed by single-crystal X-ray analysis.     

Preparation and reactivity of a novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose)

A novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose, GAF) was enzymatically prepared from 1,5-anhydro-D-fructose (1,5-AF) and cyclomaltoheptaose (beta-cyclodextrin). Cyclodextrin glucanotransferase transferred various sizes of maltooligosaccharide to 1,5-AF. Glucoamylase digested the maltooligosyl chain of the products to a glucosyl residue giving a final product, GAF. An NMR analysis of GAF elucidated that the glucose residue was linked to C-3 of the 1,5-AF residue with an ether linkage. Reactivity on the aminocarbonyl reaction of GAF with bovine serum albumin was lower than that of 1,5-AF, but was higher than that of glucose.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only d-glucose but also d-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of d-xylose metabolism, was purified, and its enzymatic properties were characterized. The purified RpXDH preferred NAD⁺ as its coenzyme and showed substrate specificity for xylitol, d-sorbitol, and ribitol. cDNA cloning of xyl2 gene encoding RpXDH revealed that the gene included a coding sequence of 1,092?bp with a molecular mass of 39,185?kDa. Expression of the xyl2 in R. pusillus NBRC 4578 was induced by d-xylose, and the expression levels were increased with accumulation of xylitol. The xyl2 gene was expressed in Escherichia coli, and coenzyme preference of the recombinant RpXDH was reversed from NAD⁺ to NADP⁺ in the double mutant D205A/I206R by site-directed mutagenesis.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

1,5-Anhydro-D-fructose; a versatile chiral building block: biochemistry and chemistry

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme alpha-(1-->4)-glucan lyase degrades starch to 1,5-anhydro-D-fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro-D-fructose is found in natural materials as a degradation product of alpha-(1-->4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro-D-fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro-D-fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6-O-acylated derivatives of 1,5-anhydro-D-fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro-D-fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro-D-fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by alpha-(1-->4)-glucan lyase, the application of 1,5-anhydro-D-fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.