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

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

doi:10.3390/ijms20225809

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 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 Vmax was determined to be 177 U mg−1 (uridine-5’-triphosphate (UTP)) and Km 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.

Highlights

Uridine-5’-diphosphate (UDP)-glucose is a key metabolite in most organisms and used in a variety of reactions of the sugar and starch metabolism, sugar interconversions, amino and nucleotide sugar metabolism, biosynthesis of antibiotics and cell envelope components, and as precursor for different primary and secondary metabolites [1,2,3]
Via a genome mining approach two genes encoding for two UDP-glucose pyrophosphorylases (GalUs) were identified in Rhodococcus opacus 1CP
Having a look at the genomic environment of both genes, various genes were found flanking RogalU1, which were involved in the sugar- and nucleotide metabolism, e.g., a mannose 6-phosphate isomerase, a D-glycero-D-manno-heptose 1-phosphate guanosyltransferase or a undecaprenyl-phosphate galactose phosphotransferase, whereas RogalU2 is flanked by cation channels, heat shock proteins or putative regulatory proteins

Summary

Introduction

Uridine-5’-diphosphate (UDP)-glucose is a key metabolite in most organisms and used in a variety of reactions of the sugar and starch metabolism, sugar interconversions, amino and nucleotide sugar metabolism, biosynthesis of antibiotics and cell envelope components, and as precursor for different primary and secondary metabolites [1,2,3]. It was presented that whole cell catalysis by sucrose synthase (SuSy) with UDP as precursor led to large-scale production of 100 gUDP-glucose/L with a yield of 86% [3]. Another report with 0.1 g/L of free SuSy could achieve 144 gUDP-glucose/L with a comparable conversion rate of 85% based on the precursor UDP [1]. The enzymatic production of various sugar-nucleotides can be realized via several UDP-glucose synthesizing enzymes (Scheme 1). Nucleotidyl transferases, like UDP-glucose pyrophosphorylase have been known for a long time but were seldom employed as biocatalysts to

Results

Discussion

Conclusion