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Uridine-5&#8217;-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 <i>Ro</i>GalU1 and <i>Ro</i>GalU2) are encoded by most <i>Rhodococcus</i> 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&#8212;<i>Rhodococcus opacus</i> 1CP&#8212;were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of <i>Ro</i>GalU2, the <i>V</i>_max was determined to be 177 U mg^&#8722;1 (uridine-5&#8217;-triphosphate (UTP)) and <i>K</i>_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 <i>Ro</i>GalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, <i>Ro</i>GalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.