Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi.

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg -ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase.

An ortho C-methylation/O-glycosylation motif on a hydroxy-coumarin scaffold, selectively installed by biocatalysis.

Various bioactive natural products, like the aminocoumarin antibiotics novobiocin and coumermycin, exhibit an aromatic C-methyl group adjacent to a glycosylated phenolic hydroxyl group. Therefore, tailoring of basic phenolic scaffolds to contain the intricate C-methyl/O-glycosyl motif is of high interest for structural and functional diversification of natural products. We demonstrate site-selective 8-C-methylation and 7-O-β-d-glucosylation of 4,5,7-trihydroxy-3-phenyl-coumarin (1) by S-adenosyl-l-methionine dependent C-methyltransferase (from Streptomyces niveus) and uridine 5'-diphosphate glucose dependent glycosyltransferase from apple (Malus × domestica).

Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage.

The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively H-labeled and deoxygenated substrates.

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase.

Nucleotide sugar-dependent ("Leloir") glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5'-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis.

Creating a Water-Soluble Resveratrol-Based Antioxidant by Site-Selective Enzymatic Glucosylation.

The phytochemical resveratrol (trans-3,5,4'-trihydroxystilbene) has drawn great interest as health-promoting food ingredient and potential therapeutic agent. However, resveratrol shows vanishingly low water solubility; this limits its uptake and complicates the development of effective therapeutic forms. Glycosylation should be useful to enhance resveratrol solubility, with the caveat that unselective attachment of sugars could destroy the molecule's antioxidant activity.

Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism.

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris.

A two-step O- to C-glycosidic bond rearrangement using complementary glycosyltransferase activities.

An efficient 2'-O- to 3'-C-β-d-glucosidic bond rearrangement on the dihydrochalcone phloretin to convert phlorizin into nothofagin was achieved by combining complementary O-glycosyltransferase (OGT) and C-glycosyltransferase (CGT) activities in a one-pot transformation containing catalytic amounts of uridine 5'-diphosphate (UDP). Two separate enzymes or a single engineered dual-specific O/CGT were applied. Overall (quantitative) conversion occurred in two steps via intermediary UDP-glucose and phloretin.

Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the -Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea.

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like -glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferase-catalyzed transformation that involves perfectly selective 3'--β-d-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. -Glucosyltransferase from (rice) was used for phloretin -glucosylation from uridine 5'-diphosphate (UDP)-glucose, which was supplied continuously through conversion of sucrose and UDP catalyzed by sucrose synthase from (soybean).

Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris.

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH.