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375719D5e77eb08eeac2f4134d International Journal of Molecular Sciences Article Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP—RoGalU1 and RoGalU2 Antje Kumpf 1,2,3,*, Anett Partzsch 1, André Pollender 1 , Isabel Bento 2 and Dirk Tischler 3,* 1 Environmental Microbiology, Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, Germany; [email protected] (A.P.); [email protected] (A.P.) 2 EMBL Hamburg, Notkestr. 85, 22607 Hamburg, Germany; [email protected] 3 Microbial Biotechnology, Faculty of Biology & Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44780 Bochum, Germany * Correspondence: [email protected] (A.K.); [email protected] (D.T.); Tel.: +49-234-32-22082 (A.K.); +49-234-32-22656 (D.T.) 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 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 1 was determined to be 177 U mg− (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. Keywords: glycosylation; UDP-glucose pyrophosphorylase; UDP-glucose; nucleotide donors; Rhodococcus, Actinobacteria, gene redundancy; Leloir glycosyltransferases; activated sugar; UTP 1. Introduction Uridine-5’-diphosphate (UDP)-glucose is a key metabolite in most organisms and thus 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 [1–3]. Being an interesting and valuable compound, UDP-glucose attracted more and more attention for biotechnological applications. As an example, in glycosylation reactions, their production and application in fine chemical scale could be shown [1–3]. 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 (Scheme1). Glycosyltransferases, like sucrose synthase, are the most common used representatives [1,4–7]. Nucleotidyl transferases, like UDP-glucose pyrophosphorylase have been known for a long time but were seldom employed as biocatalysts to Int. J. Mol. Sci. 2019, 20, 5809; doi:10.3390/ijms20225809 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5809 2 of 23 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 25 produce UDP-glucose,UDP-glucose, maybemaybe due due to to low low specific specific activities. activities. Those Those enzymes enzymes are are more more often often studied studied for theirfor their metabolic metabolic role. role. Scheme 1. Reactions in which UDP-glucose is consumed or formed. UDP: Uridine-5’-diphosphate, UMP:Scheme Uridine-5’-monophosphate, 1. Reactions in which UDP-glucose UTP: Uridine-5’-triphosphate, is consumed or formed. NAD+ /UDP:NADH: Uridine-5’-diphosphate, Nicotinamide adenine + dinucleotideUMP: Uridine-5’-monophosphate, in the oxidized and reduced UTP: form. Uridine-5’-triphosphate, NAD /NADH: Nicotinamide adenine dinucleotide in the oxidized and reduced form. UDP-glucose pyrophosphorylases (GalU or UGPase; EC 2.7.7.9) catalyze the reversible reaction of glucoseUDP-glucose 1-phosphate pyrophosphorylases and UTP into UDP-glucose (GalU or andUGPase; inorganic EC 2.7.7.9) pyrophosphate catalyze the (PP reversiblei). Enzymes reaction of the GalUof glucose family 1-phosphate are ubiquitous and and UTP can into be UDP-glucose found among and the treeinorganic of life [pyrophosphate8]. With respect (PP to bacteriai). Enzymes mostly of proteobacterialthe GalU family GalUs are ubiquitous have been and studied. can be Like found many amon otherg nucleotidylthe tree of life transferases, [8]. With respect also GalU to requiresbacteria divalentmostly proteobacterial cations to promote GalUs the have reaction been studied. (Scheme Li2ke). Inmany most other cases nucleotidyl magnesium transferases, ions are employed, also GalU andrequires so far divalent only magnesium cations to chloridepromote has the been reaction investigated (Scheme [ 92).–16 In]. Themost reaction cases magnesium mechanism ions follows are aemployed, sequential and bi-bi-mechanism so far only magnesium starting chloride with the has binding been investigated of UTP to the[9–16]. active The site, reaction in presence mechanism of a magnesiumfollows a sequential ion [17], bi-bi-mechanism followed by the bindingstarting with of glucose the binding 1-phosphate. of UTP Theto the octahedral active site, coordination in presence sphereof a magnesium of the magnesium ion [17], positions followed the substratesby the bind in theing right of wayglucose and enables1-phosphate. the nucleophilic The octahedral attack ofcoordination glucose 1-phosphate sphere of ontheUTP magnesium [18]. A lysine,positions an aspartatethe substrates and several in the waterright way molecules and enables within thethe activenucleophilic site help attack to stabilize of glucose the position 1-phosphate of the substrateson UTP [18]. and A cofactor lysine, foran theaspartate proper nucleophilicand several attackwater ofmolecules the phosphoryl within the oxygen active of site glucose help 1-phosphate to stabilize towardsthe position the αof-phosphor the substrates atom and of UTP cofactor [17]. Finally,for the 2+ PPproperi is released nucleophilic from attack the GalU of the/Mg phosphoryl/UDP-glucose oxygen complex of glucose [17]. 1-phosphate towards the α-phosphor atomGalU of UTP enzymes [17]. Finally, have been PPi shown is released to play from crucial the rolesGalU/Mg in galactose2+/UDP-glucose fermentation complex [19]. Other[17]. metabolic pathwaysGalU inenzymes which thishave enzyme been shown family to is activeplay crucia are similarl roles likein galactose the ones UDP-glucosefermentation itself[19]. Other plays anmetabolic important pathways role. Itin produces which this UDP-glucose enzyme family as is precursor active are for similar sucrose, like glucanthe ones and UDP-glucose amylose in itself the sugarplays an and important starch metabolism. role. It produces Furthermore, UDP-glucose it plays as a roleprecursor in galactose for sucrose, metabolism glucan and and glucuronate amylose in interconversion,the sugar and starch where galactosemetabolism. 1-phosphate Furthermore, is over it allplays converted a role intoin galactose UDP-glucuronate metabolism with and the helpglucuronate of a hexose interconversion, 1-phosphate uridylylwhere galactose transferase 1-phosphate and a UDP-glucose is over 6-dehydrogenase.all converted into In aminoUDP- sugarglucuronate and nucleotide with the sugar help metabolism, of a hexose GalU 1-phosphate is active in uridylyl interconverting transferase different and UDP-sugarsa UDP-glucose via the6- intermediatedehydrogenase. UDP-glucose. In amino sugar Additionally, and nucleotide it can besugar found metabolism, in glycolipid GalU metabolism is active in and interconverting the synthesis different UDP-sugars via the intermediate UDP-glucose. Additionally, it can be found in glycolipid metabolism and the synthesis of antibiotics. Respectively, many GalU enzymes from several Int. J. Mol. Sci. 2019, 20, 5809 3 of 23 Int.of antibiotics.J. Mol. Sci. 2019 Respectively,, 20, x FOR PEER manyREVIEW GalU enzymes from several organisms have been investigated3 of 25 to date, e.g., GalUs of plants [20–24], of mammals [25–27], including human [28], parasites [29], organismsand many differenthave been bacteria investigated [9,10, 12to ,date,13,30 e.g.–33 ],GalUs only of to plants name a[20–24], few groups. of mammals Fields [25–27], of study including included humanthe determination [28], parasites of structures[29], and many [17,30 different,34], biochemical bacteria [9,10,12,13,30–33], characterization [only14,31 to,35 name,36] and a few biological groups. Fieldsfunction of [15study,33,37 included]. the determination of structures [17,30,34], biochemical characterization [14,31,35,36] and biological function [15,33,37]. Scheme 2. ProposedProposed reaction reaction mechanism mechanism of ofUDP-glucose UDP-glucose pyro pyrophosphorylase,phosphorylase, adapted adapted
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