Jürgen Seibel

Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer.

Industrial carbohydrate biotransformations

Nearly all major industrial processes which involve carbohydrates, include biotechnological transformations. This is due to the complex nature of carbohydrates where stereo- and regioselectivity are highly complex and difficult to control. Enzymes and microorganisms work highly selectively and efficiently in water solution, and provide high yield in general. The article focuses on different types of reactions, including large-scale processes. Topics are hydrolytic reactions, including starch processing, oxidation and reduction transformations including organic acids, such as gluconic and ketogluconic acids and vitamin C synthesis, and isomerization and transfer reactions, which are established on a very large scale to produce glucose/fructose syrups and sucrose isomers. The article will further discuss some mechanistic aspects which are relevant for technology and present selected details of industrial-scale processing. Finally an outlook outlines perspectives of future processes.

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.

Insights into polymer versus oligosaccharide synthesis : mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium

A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis, the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterized kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram-positive Bacillus subtilis and Gram-negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg370 and Asn252 were of high relevance in polymer compared with oligosaccharide synthesis.

Glycosylation with activated sugars using glycosyltransferases and transglycosidases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a requirement for the general availability of larger amounts of varying carbohydrate structures. Thus the synthesis of oligo-, polysaccharides and glycosylated substances/products represents a major challenge. Activated sugars are key substrates for synthesis and glycosylation. Nucleotide activated sugars are natural tools for highly selective synthesis, providing complex polysaccharides, glycopeptides, glycolipids etc. However their high cost and availability limit their application. Sucrose acts as an activated substrate for a range of sucrase enzymes elaborating natural polysacchrides of the glucan and fructan type, which also serve for the synthesis and technical production of different oligosaccharides. Sucrose analogues have been shown to extend the range of oligosaccharide synthesis making new structures available incorporating further monosaccharides, such as mannose, galactose, xylose, rhamnose and fucose. A short overview of the use of glycosyl phosphates and glycosyl fluorides as substrates is also given.

Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: Toward tailor-made polymer and oligosaccharide products

Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency.

Synthesis of novel fructooligosaccharides by substrate and enzyme engineering.

Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its beneficial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides by substrate and enzyme engineering. Using an engineered levansucrase enzyme (SacB of Bacillus subtilis), and sucrose analogues (alpha-Xyl-1,2-beta-Fru or alpha-Gal-1,2-beta-Fru), the product profile shifted from the fructan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides (galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, for the synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to produce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabled in combination with the sucrose analogue Xyl-Fru for the first time the direct synthesis of a 6-kestose analogue (alpha-Xyl-1,2-beta-Fru-2,6-beta-Fru). The different glycopyranosyl-residues (i.e. galactose and xylose) that cap fructooligosaccharides may alter prebiotic and biochemical properties.

Identification of new acceptor specificities of glycosyltransferase R with the aid of substrate microarrays.

Finding opportunities to construct sugar motifs and to transfer them to targets of biological relevance and rapid identification of glycosylation events are important goals for glycobiology and a field of increasing interest. Here we have applied an enzyme microarray screening system for the identification of new acceptor specificities of the glycosyltransferase R (GTFR) from Streptococcus oralis (E.C. 2.4.1.5), which was able to effect the synthesis of sugar motifs in short times and with low amounts of substrate. These observations resulted in the development of a convenient α‐glycosylation by the non‐Leloir glycosyltransferase GTFR, with sucrose as substrate and with different alcohols and amino acid derivatives as acceptors, for the synthesis of glycoethers and glycosylated amino acids not observed with the use of familiar GTFs with high sequence homology.

Tailor-made fructooligosaccharides by a combination of substrate and genetic engineering.

The combination of sucrose analogues as novel substrates (substrate engineering) and highly active recombinant β‐fructofuranosidase from A. niger (genetic engineering) provides a new powerful tool for the efficient preparative synthesis of tailor‐made saccharides of the important 1‐kestose and 1‐nystose type headed with different monosaccharides of interest. These novel compounds have been isolated. They did not display toxic effects or suppress cell growth in initial studies, making these new compounds potential candidates for prebiotics.

Super‐Resolution Imaging of Plasma Membrane Glycans

Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.