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

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.

[1]  S. R. Marsden,et al.  Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach , 2019, International journal of molecular sciences.

[2]  S. R. Marsden,et al.  Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability , 2019, Applied and Environmental Microbiology.

[3]  J. Tokas,et al.  UDP-glucose pyrophosphorylase: Isolation, purification and characterization from developing thermotolerant wheat (Triticum aestivum) grains. , 2018, Protein expression and purification.

[4]  A. Pratap,et al.  Production and Quantitative Analysis of Trehalose Lipid Biosurfactants Using High-Performance Liquid Chromatography , 2018, Journal of Surfactants and Detergents.

[5]  Sudhir Kumar,et al.  MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. , 2018, Molecular biology and evolution.

[6]  Torsten Schwede,et al.  SWISS-MODEL: homology modelling of protein structures and complexes , 2018, Nucleic Acids Res..

[7]  B. Kobe,et al.  Crystal structure and insights into the oligomeric state of UDP-glucose pyrophosphorylase from sugarcane , 2018, PloS one.

[8]  M. Rejžek,et al.  Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity. , 2017, Biochimica et biophysica acta. Proteins and proteomics.

[9]  Peiyuan Yao,et al.  Exploiting the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis and its application in synthesis of glycosides. , 2017, Journal of biotechnology.

[10]  Bernd Nidetzky,et al.  Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase. , 2017, Biotechnology and bioengineering.

[11]  Y. Kawarabayasi,et al.  Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position , 2016, Applied and Environmental Microbiology.

[12]  B. Nidetzky,et al.  Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5′‐Diphosphate‐Glucose by Sucrose Synthase , 2016 .

[13]  B. Nidetzky,et al.  Sequence determinants of nucleotide binding in Sucrose Synthase: improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues , 2016, Protein engineering, design & selection : PEDS.

[14]  B. Nidetzky,et al.  Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development. , 2016, Biotechnology advances.

[15]  A. Ebrecht,et al.  On the Ancestral UDP-Glucose Pyrophosphorylase Activity of GalF from Escherichia coli , 2015, Front. Microbiol..

[16]  F. Cañada,et al.  Structure and Function of Prokaryotic UDP-Glucose Pyrophosphorylase, A Drug Target Candidate. , 2015, Current medicinal chemistry.

[17]  J. Schneider,et al.  A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism , 2015, Scientific Reports.

[18]  S. Bornemann,et al.  Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis , 2015, Biochimica et biophysica acta.

[19]  D. Eulberg,et al.  Gene redundancy of two-component (chloro)phenol hydroxylases in Rhodococcus opacus 1CP. , 2014, FEMS microbiology letters.

[20]  S. Benini,et al.  Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora. , 2014, Acta crystallographica. Section F, Structural biology communications.

[21]  M. Ihara,et al.  Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120. , 2014, Journal of bioscience and bioengineering.

[22]  Xavier Robert,et al.  Deciphering key features in protein structures with the new ENDscript server , 2014, Nucleic Acids Res..

[23]  Wim Soetaert,et al.  Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases , 2013, Applied and Environmental Microbiology.

[24]  B. Nidetzky,et al.  Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea , 2013, Advanced synthesis & catalysis.

[25]  M. Schlömann,et al.  Trehalose phosphate synthases OtsA1 and OtsA2 of Rhodococcus opacus 1CP. , 2013, FEMS microbiology letters.

[26]  M. Schlömann,et al.  Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme , 2012, Applied and Environmental Microbiology.

[27]  M. Schlömann,et al.  One-Component Styrene Monooxygenases: An Evolutionary View on a Rare Class of Flavoproteins , 2012, Applied Biochemistry and Biotechnology.

[28]  A. Iglesias,et al.  Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor , 2011, Journal of bacteriology.

[29]  Chengping Lu,et al.  Molecular cloning and analysis of the UDP-Glucose Pyrophosphorylase in Streptococcus equi subsp. zooepidemicus , 2011, Molecular Biology Reports.

[30]  E. Schulz,et al.  Structural basis for the broad substrate range of the UDP-sugar pyrophosphorylase from Leishmania major. , 2011, Journal of molecular biology.

[31]  M. Quail,et al.  The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions , 2010, PLoS genetics.

[32]  Jongkeun Choi,et al.  Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase , 2010, Molecules and cells.

[33]  L. Kleczkowski,et al.  Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase. , 2009, Biochimica et biophysica acta.

[34]  W. V. van Berkel,et al.  Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP , 2009, Journal of bacteriology.

[35]  M. Hirai,et al.  A Chloroplastic UDP-Glucose Pyrophosphorylase from Arabidopsis Is the Committed Enzyme for the First Step of Sulfolipid Biosynthesis[W][OA] , 2009, The Plant Cell Online.

[36]  A. Iglesias,et al.  UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation. , 2009, Biochimie.

[37]  Honghai Wang,et al.  Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase. , 2008, Protein expression and purification.

[38]  L. Kleczkowski,et al.  Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis. , 2008, Biochimica et biophysica acta.

[39]  Sanjay Kumar Gupta,et al.  Regulation of UDP-glucose pyrophosphorylase isozyme UGP5 associated with cold-sweetening resistance in potatoes. , 2008, Journal of plant physiology.

[40]  R. Mizanur,et al.  A thermostable promiscuous glucose-1-phosphate uridyltransferase from Helicobacter pylori for the synthesis of nucleotide sugars , 2008 .

[41]  J. Thoden,et al.  Active site geometry of glucose‐1‐phosphate uridylyltransferase , 2007, Protein science : a publication of the Protein Society.

[42]  R. Huber,et al.  Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major* , 2007, Journal of Biological Chemistry.

[43]  E. Mitchell,et al.  The Complex of Sphingomonas elodea ATCC 31461 Glucose-1-Phosphate Uridylyltransferase with Glucose-1-Phosphate Reveals a Novel Quaternary Structure, Unique among Nucleoside Diphosphate-Sugar Pyrophosphorylase Members , 2007, Journal of bacteriology.

[44]  Lili Zhu,et al.  Multiple isoforms of UDP-glucose pyrophosphorylase in rice , 2007 .

[45]  J. Thoden,et al.  The molecular architecture of glucose‐1‐phosphate uridylyltransferase , 2007, Protein science : a publication of the Protein Society.

[46]  George N Phillips,et al.  Structure and dynamics of UDP-glucose pyrophosphorylase from Arabidopsis thaliana with bound UDP-glucose and UTP. , 2007, Journal of molecular biology.

[47]  A. Bracher,et al.  Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae. , 2006, Journal of molecular biology.

[48]  E. Mitchell,et al.  Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate. , 2006, Acta Crystallographica. Section F : Structural Biology and Crystallization Communications.

[49]  A. Fialho,et al.  Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities , 2005, Applied and Environmental Microbiology.

[50]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.

[51]  Y. Kawarabayasi,et al.  Identification of an Extremely Thermostable Enzyme with Dual Sugar-1-phosphate Nucleotidylyltransferase Activities from an Acidothermophilic Archaeon, Sulfolobus tokodaii strain 7* , 2005, Journal of Biological Chemistry.

[52]  E. Barrett,et al.  Effects of Insulin and Transgenic Overexpression of UDP-glucose Pyrophosphorylase on UDP-glucose and Glycogen Accumulation in Skeletal Muscle Fibers* , 2005, Journal of Biological Chemistry.

[53]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[54]  E. Agosin,et al.  Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum , 2004, Applied and Environmental Microbiology.

[55]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[56]  I. Rayment,et al.  Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism* , 2003, Journal of Biological Chemistry.

[57]  A. Fialho,et al.  Characterization of the ugpG gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer Sphingomonas paucimobilis ATCC 31461 , 2003, Molecular Genetics and Genomics.

[58]  O. V. Moiseeva,et al.  A New Modified ortho Cleavage Pathway of 3-Chlorocatechol Degradation by Rhodococcus opacus 1CP: Genetic and Biochemical Evidence , 2002, Journal of bacteriology.

[59]  L. De Vuyst,et al.  Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03 , 2000, Applied and Environmental Microbiology.

[60]  Yu Sam Kim,et al.  Cloning, Sequencing, and Expression of UDP-Glucose Pyrophosphorylase Gene from Acetobacter xylinum BRC5 , 2000, Bioscience, biotechnology, and biochemistry.

[61]  Se Yong Lee,et al.  Biochemical characterization of a UDP‐sugar pyrophosphorylase from Thermus caldophilus GK24 , 1999, Biotechnology and applied biochemistry.

[62]  L. Kleczkowski Glucose activation and metabolism through UDP-glucose pyrophosphorylase in plants , 1994 .

[63]  E. P. Kennedy,et al.  UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme , 1994, Journal of bacteriology.

[64]  P. Reeves,et al.  Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidylyl-transferase from the cloned rfbA gene. , 1993, European journal of biochemistry.

[65]  William R. Taylor,et al.  The rapid generation of mutation data matrices from protein sequences , 1992, Comput. Appl. Biosci..

[66]  S. Enfors,et al.  Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli , 1991, Applied and environmental microbiology.

[67]  Dutta Sk UDPglucose pyrophosphorylase from Ehrlich ascites carcinoma cell--purification and characterization. , 1985 .

[68]  C. Granzow,et al.  Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettre ascites tumor cells , 1981, The Journal of cell biology.

[69]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[70]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[71]  Toshimasa Yano,et al.  Dynamic behavior of the chemostat subject to substrate inhibition , 1969 .

[72]  S. Flitsch,et al.  Discovery and Biochemical Characterization of a Thermostable Glucose-1-phosphate Nucleotidyltransferase from Thermodesulfatator indicus. , 2017, Protein and peptide letters.

[73]  A. Ebrecht,et al.  The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate. , 2015, Biochimica et biophysica acta.

[74]  P. Loppnau,et al.  A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment. , 2012, Protein expression and purification.

[75]  S. K. Dutta UDPglucose pyrophosphorylase from Ehrlich ascites carcinoma cell--purification and characterization. , 1985, Indian journal of biochemistry & biophysics.

[76]  P. Robbins,et al.  CONTROL ASPECTS OF URIDINE 5'-DIPHOSPHATE GLUCOSE AND THYMIDINE 5'-DIPHOSPHATE GLUCOSE SYNTHESIS BY MICROBIAL ENZYMES. , 1965, The Journal of biological chemistry.