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

• 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 \(\it Ro\)GalU1 and \(\it Ro\)GalU2) are encoded by most \(\it Rhodococcus\) 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—\(\textit {Rhodococcus opacus}\) 1CP—were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of \(\it Ro\)GalU2, the \(\it {V}_{max}\) 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 \(\it Ro\)GalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, \(\it Ro\)GalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.