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Open AccessArticle

Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP—RoGalU1 and RoGalU2

by Antje Kumpf, Anett Partzsch, André Pollender, Isabel Bento and Dirk Tischler

Int. J. Mol. Sci. 2019, 20(22), 5809; https://doi.org/10.3390/ijms20225809 - 19 Nov 2019

Cited by 8 | Viewed by 5957

Uridine-5’-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism—Rhodococcus opacus 1CP—were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the V_max was determined to be 177 U mg⁻¹ (uridine-5’-triphosphate (UTP)) and K_m to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products)

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36 pages, 6771 KB

Open AccessReview

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

by Luuk Mestrom, Marta Przypis, Daria Kowalczykiewicz, André Pollender, Antje Kumpf, Stefan R. Marsden, Isabel Bento, Andrzej B. Jarzębski, Katarzyna Szymańska, Arkadiusz Chruściel, Dirk Tischler, Rob Schoevaart, Ulf Hanefeld and Peter-Leon Hagedoorn

Int. J. Mol. Sci. 2019, 20(21), 5263; https://doi.org/10.3390/ijms20215263 - 23 Oct 2019

Cited by 92 | Viewed by 12190

Abstract

Enzymes are nature’s catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations. Full article

(This article belongs to the Special Issue Carbohydrate-Active Enzymes: Structure, Activity and Reaction Products)

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21 pages, 1707 KB

Open AccessFeature PaperArticle

Enzymatic Cascades for Tailored ¹³C₆ and ¹⁵N Enriched Human Milk Oligosaccharides

by Thomas Fischöder, Samanta Cajic, Valerian Grote, Raphael Heinzler, Udo Reichl, Matthias Franzreb, Erdmann Rapp and Lothar Elling

Molecules 2019, 24(19), 3482; https://doi.org/10.3390/molecules24193482 - 25 Sep 2019

Cited by 8 | Viewed by 4981

Abstract

Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple “routine” method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [¹³C₆]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[¹⁵N]GlcNAc and its application for the enzymatic synthesis of [¹³C₆/¹⁵N]lacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations. Full article

(This article belongs to the Section Bioorganic Chemistry)

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