Jan Marcussen

α-1,4-Glucan lyases producing 1,5-anhydro-d-fructose from starch and glycogen have sequence similarity to α-glucosidases

Abstract In the past few years a novel enzyme α-1,4-glucan lyase (EC 4.2.2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known α-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75–80% among the α-1,4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal α-1,4-glucan lyases is only 25–28%. Notably database searches disclosed that the α-1,4-glucan lyases have a clear identity of 23–28% with α-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and α-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in α-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of α-1,4-glucan lyases with α-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

A General Method Based on the Use of N-Bromosuccinimide for Removal of the Thiophenyl Group at the Anomeric Position to Generate A Reducing Sugar with the Original Protecting Groups Still Present

Abstract Efficient conversion of a range of different phenyl thioglycosides into their hemiacetals has been achieved by treatment with N-bromosuccinimide in aqueous acetone. The method is mild and general since it does not interfere with the presence of other protecting groups like acetate, benzyl, benzylidene acetal, tert-butyldiphenylsilyl groups, and the O-glycosidic bond (e.g. di-, tetra-, and pentasaccharide thioglycosides).

Methods for the assay of 1,5-anhydro-d-fructose and α-1,4-glucan lyase

Abstract 1,5-Anhydro- d - arabino -hex-2-ulose (1,5-anhydro- d -fructose, 1,5AnFru), produced by α-1,4-glucan lyase (EC 4.2.2.13) acting on starch, glycogen, or related d -glucose oligo- and polysaccharides as substrate, reacts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room temperature (22 °C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5AnFru content in the range of 0.5 to 16 μmol (80 μg to 2.6 mg) mL −1 . 1,5AnFru determined by this colorimetric, one test tube one reagent method, was in good agreement with that found by 1 H-NMR spectroscopy and HPLC. The DNS method is also specific as other reducing sugars, such as glucose, maltose maltosaccharides, starch and glycogen do not give a colour with DNS at 22 °C; therefore, they do not interfere in the determination. The DNS method is applicable to lyase assay for both cell-free extracts and purified enzyme. Methods for reducing sugar analyses, based on the reduction of ferric and cupric ions, were examined for 1,5AnFru and they proved to be quantitative but in contrast to the DNS method, they were not specific. Instead of assaying 1,5AnFru, the activity of α-1,4-glucan lyase was analysed enzymatically by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl α-maltopentaoside as substrate, respectively.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : I. Spatial distribution of endogenous cytokinins in relation to leaf growth.

Immunoaffinity purification of zeatin, dihydrozeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adenosine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydrozeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spatial distribution of endogenous cytokinins in rapidly expanding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly expanding leaves was found to be correlated with the rate of leaf expansion which is high (∼40%/24 h) in the basal leaf tissue and low (∼10%/24 h) near the leaf tip. Applied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.

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.

Efficient purification, characterization and partial amino acid sequencing of two α-1,4-glucan lyases from fungi

alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.

Chemical synthesis and NMR spectra of a protected branched-tetrasaccharide thioglycoside, a useful intermediate for the synthesis of branched oligosaccharides

Acid-catalyzed thiophenolysis of per-O-acetylated 1,6-anhydromaltose (3) gave phenyl 2,3-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-alpha-D- glucopyranosyl)-1-thio-beta-D-glucopyranoside (4) in quantitative yield. Phenyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside (5) was obtained by acid-catalyzed thiophenolysis of maltose octaacetate (2), using trimethylsilyl triflate as catalyst, and subsequent deacetylation. Standard benzylation of 5 gave phenyl 2,3-di-O-benzyl-4-O- (2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-1-thio-beta-D-glucopy ran oside (6) which upon treatment with N-bromosuccinimide in aqueous acetone gave 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-D-glucopyranose (8). Compound 8 was treated with trichloroacetonitrile in the presence of anhydrous potassium carbonate to give 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl- alpha-D-glucopyranosyl) -alpha,beta-D-glucopyranosyl trichloroacetimidate (9), which was effectively used as the glycosyl donor in the condensation reaction with compound 4, using trimethylsilyl triflate as catalyst, to obtain the branched tetrasaccharides phenyl O-[2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)- (1-->4)]-O-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->6)-O-(2,3,4, 6- tetra-O-acetyl-alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-acetyl-1-thio-be ta-D- glucopyranoside (10) and phenyl O-[(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)]- O-(2,3,4-tri-O-benzyl-beta-D-glucopyranosyl)-(1-->6)-O-(2,3,4,6-tetra-O- acetyl- alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-acetyl-1-thio-beta-D-glucopy ran oside (11) in 67 and 21% yield, respectively. A complete NMR interpretation of 10 is presented. Alternative methodologies for the synthesis of the branched tetrasaccharides were investigated. Chemical synthesis of the phenyl thioglycoside 5 was achieved by deacetylation of 4. Reaction of 6 with diethylaminosulfur trifluoride in the presence of N-bromosuccinimide gave 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-alpha,beta-D-glucopyranosyl fluoride (7) in 78% yield. Subsequent condensation of 7 and 4, using the combination silver perchlorate-stannous chloride as catalyst, gave the corresponding branched tetrasaccharides 10 and 11 in 55 and 10% yield, respectively.

Isolation and expression of two cDNA clones encoding UDP-galactose epimerase expressed in developing seeds of the endospermous legume guar

Abstract UDP-galactose 4′-epimerase (UDPG epimerase) catalyses the reversible conversion of UDP- d -glucose to UDP- d -galactose. This compound is a precursor for the biosynthesis of various galactosides and cell wall polymers, including galactomannan which is the main storage polysaccharide in endospermous legumes. Using functional complementation of a UDPG epimerase deficient Escherichia coli mutant (PL-2) by a cDNA expression library from immature guar ( Cyamopsis tetragonoloba ) seeds, galactose metabolising colonies with UDPG epimerase activities comparable to wild type level were obtained. Two cDNA clones (GEPI42 and GEPI48) encoding two different UDPG epimerases were isolated. Re-transformation of PL-2 by plasmid DNA, isolated from either of the two clones, resulted in numerous galactose-metabolising colonies, all with high UDPG epimerase activities. GEPI42 and GEPI48 encoded proteins with 354 and 350 amino acid residues, respectively, corresponding to deduced molecular weights of 39 286 and 38 373 Dalton, respectively. The amino acid sequence identity was 66.9%. Southern analysis of genomic guar DNA confirmed the origin and distinctness of the two UDPG epimerase genes. Analysis by immunohistochemistry showed the presence of significant levels of UDPG epimerase antigen in the endosperm of immature seeds with rapid galactomannan biosynthesis. In the endosperm of seeds close to maturity where galactomannan deposition has ceased, no antigen was detected. These data indicate that one or both of the two cloned UDPG epimerase genes are expressed in guar endosperm at a developmental stage where galactomannan biosynthesis occurs, suggesting that one or both may be involved in this process.

A group of α-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.

Structure of 1,5-Anhydro-D-Fructose: X-ray Analysis of Crystalline Acetylated Dimeric Forms

Abstract Acetylation of 1,5-anhydro-D-fructose under acidic conditions gave two crystalline acetylated dimeric forms, which by X-ray analysis were shown to be diastereomeric spiroketals formed between C-2 and C-2/C-3. The structures of the compounds differed only at the configuration at C-2. Acetylation or benzoylation of 1,5-anhydro-D-fructose in pyridine yielded 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enos-2-ulopyra-nos or crystalline 1,5-anhydro-3,6-di-O-benzoyl-4-deoxy-D-glycero-hex-3-enos-2-ulo-pyranose. 1. Presented at the 9th European Carbohydrate Symposium, Utrecht, Netherlands, July 6-1 1, 1997, Poster A138.