**Antje Kumpf 1,2,3,\*, Anett Partzsch 1, André Pollender 1, Isabel Bento <sup>2</sup> and Dirk Tischler 3,\***


Received: 28 October 2019; Accepted: 16 November 2019; Published: 19 November 2019

**Abstract:** 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 *Ro*GalU1 and *Ro*GalU2) 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 *Ro*GalU2, the *V*max was determined to be 177 U mg−<sup>1</sup> (uridine-5'-triphosphate (UTP)) and *K*<sup>m</sup> 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 *Ro*GalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, *Ro*GalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.

**Keywords:** glycosylation; UDP-glucose pyrophosphorylase; UDP-glucose; nucleotide donors; *Rhodococcus*, Actinobacteria, gene redundancy; Leloir glycosyltransferases; activated sugar; UTP
