Cloning, Expression, and Characterization of UDP-glucose Pyrophosphorylase from Sphingomonas chungbukensis DJ77

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Soc. , 30, No. 6 Moon-Young

Cloning, Expression, and Characterization of UDP-glucose Pyrophosphorylase from Sphingomonas chungbukensis DJ77

Moon-Young Yoon, Kyoung-jin Lee,Hea-ChulPark, Sung-Ha Park,' Sang GonKim,^* Sung-Kun Kim,* and Jung-Do Choi',*

Department ofChemistry, Research Institutefor Natural Sciences, Hanyang University, Seoul 133-791, Korea

‘Department ofBiochemistry, Chungbuk National University, Cheogju361-763, Korea

*E-mail: jdchoi@cbucc.chungbuk.ac.kr

^Department of Chemistryand Biochemistry andInstitute of Biomedical Studies, Baylor University, Waco, Texas 76798-7348, USA

Received March 25, 2009, AcceptedMay 1, 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield.

Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates Km = 1.14 mM and V^max= 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1 -phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1 -phosphate binding site, indicates that substitution of the charge -reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

Key Words: UDP-glucosepyrophosphorylase,Sphingomonaschungbukensis, Extracellular polysaccharide, Site-directedmutagenesis

Introduction

The bacterium Sphingomonas chungbukensisDJ77 (pre viously identified as a Pseudomonas species)1 has been isolated from contaminated freshwater sediment in Daejeon, Korea.2 S. chungbukensisDJ77 has been shownto produce large quantities of extracellular polysaccharide (EPS)gellan, which can be usedas a gelling agent for foodand pharmaceutical use.3 TheEPS gellan is a linear tetrasaccharide composed of repeatingunits of 卩-D-glucuronic acid,卩-D-glucose, and a- L-rhamnose4in a 2:1:1 ratio. TheEPS biosyntheticpathway has beenpartiallyelucidated;5,6 however, furtherinvestigation isneeded to completely delineatethepathway.Thus cloning, expressing and characterizing the genes involved in EPS gellanbiosynthesis is critical for metabolicengineering of the pathway.

Uridine diphosphate (UDP)-glucosepyrophosphorylase, also termed glucose-1-phosphate uridylyltransferase (UGP, EC 2.7.7.9)is a cytosolicenzyme thatcatalyzes thereversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose-1 -phosphate. In addition, this enzyme is thoughtto catalyzethe reversibleconversion of galactose to glucose by way of the Leloir pathway.7 The ugp gene deficiencyexperiments in E. coli supportthe hypothesisthat theenzymealsoconvertsgalactoseto glucose.8UGP activity

has been foundin bothprokaryotes and eukaryotes, although the prokaryotic and eukaryotic enzymes differsignificantly.

In prokaryotes, UGP is essential for the biosynthesis of several polysaccharides suchas EPS, lipopolysaccharides, andcapsular polysaccharides.9,10 The enzymeproduct, UDP-glucose, has beenknown tobe a precursor for thesynthesis of the tetrasac- charidic unit that isinvolved in EPS biosynthesis11 and so it is ofkeen interest to investigatethe cloning and characterization of theugpgene in thisbacterium.

Inthis work,we report the cloning andexpression of the ugp gene and purificationofUGP from S. chungbukensis DJ77. Amino acidalignment, kinetic analysis and site-directed mutagenesisexperimentswere carried out tocharacterizethe enzyme.

ExperimentalSection

Materi지s. S. chungubkensisDJ77 and itsfosmid library were preparedin thelaboratory ofDr. Young-ChangKim.2 Fosmid library construction was carried out according to methods described previously,12,13 using CopyControlTM Fosmid Library Production Kit (Epicentre, Madison, USA).

Therestriction enzymesand T4 ligase were purchased from Roche (Mannheim, Germany), while Taq polymerase and other components forPCR were purchased from BioneerInc.

Abbreviations: EPS, extracellular polysaccharide; NADP⁺, nicotinamide adenine dinucleotide phosphate; UGP, UDP-glucose pyrophos- photase; IPTG, isopropyl thio-beta-D-galactoside; PCR, polymerase chain reaction; pl, isoelectric point; SDS, sodium dodecyl sulfate;

PAGE, polyacrylamide gel electrophoresis

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(Daejeon, Korea). PCR primers weresynthesized byGenotech (Daejeon, Korea).

Gene selection. Based onthe S.chungubkensisDJ77 geno

micdatabase (http://bioinfo.chungbuk.ac.kr), we found a cu292 fosmidclone that contains the ugp gene. Using the fosmid clone,we determined thewhole genesequence with the primer walking method. The reverse primers used were: 5’-GGTCA- CGAAGATCAGATGCT-3 ’, 5’-GAGATCCAGCTGACCG- ATG-3 ’，5’ -GTCTGAAACGGGGTAAGCCA-3’，and 5’ -TG- TCACTGCGCATCACGTTG-3 ’.

Constiuctionof expression vectors. PCR amplification was done using the followingforward primers: (5’-AAGGAT- CCCATGTCCTACAAACCGATTC-3’)and (5’-TTGGAT- CCTCAGGCCGCGGCGGCAAT-3’)，wheretheunderlined sequencesindicate the BamHI restriction site. Genomic DNA was used asa template for PCR. The BamH I-digestedPCR products were inserted into the pET-15b expressionvector using T4 DNA ligase. Finally, E. coli BL21(DE3)pLysS was transformed with the cloned plasmid. The sequence of the insertwasconfirmed byDNAsequencinginboth directions.

Expression and purification of recombinant protein. The transformed E. colistrains BL21(DE3)pLysS were grownat 37 oC in LBmedium containing50mg/Lampicillin, until the optical density of the culture at 600 nm reached 0.6. The inducer IPTG was added to thecultureat a final concentration of0.1 mM,followedby growth for 5 h at 30 oC. Cellswere harvested by centrifugationat 5,000 g for 20 min. The cell pelletwas resuspended in buffer (50 mM Tris, pH 8.0, 500 mM NaCl). The cell suspension waslysedby sonicationon ice, and thesoluble fraction was obtained bycentrifugationat 20,000g for 30 min. The supernatant was applied to a GE HP high trap nickel affinity column. The protein purification procedurewasconductedasdescribedbythe manufacturer’s protocol provided fromQiagen, and the purifiedprotein was analyzedon SDS-PAGE. Native-gel electrophoresis was done, with methods described previously.14 Protein concentration was estimated by the Bradford method,15 using a bovine serumalbuminstandard.

Enzyme assay. The activity of UGP was assayed by previously described methods.16,17TheUGP-catalyzed glucose- 1-phosphate was converted to glucose-6-phosphate by the enzyme phosphoglucomutase (purchased from Roche Co.).

The glucose-6-phosphate was then used as substrate for glucose-6-phosphate dehydrogenase(purchased from Roche Co.) with NADP+ used as a cofactor. The UGP activity was monitoredviathe increase of NADPHconcentrationbythe reduction ofNADP+, and the absorbance at 340 nm was detected usinga BeckmanDU-650 spectrophotometer at 30 oC. The standard reaction mixture for UGP activity assay contained100 mMTris-HCl (pH8.5),2 mM pyrophosphate,10 mM MgCl2, 10 mMNADP+, 1 unit of phosphoglucomutase, and1 unit ofglucose-6-phosphate dehydrogenase,along with 0.05 - 1.2 mM UDP-glucose.14,18-20 To study the effect of changingpH, 100 mMMES (pH 5.5 - 6.5), 100 mM MOPS (pH6.5-8.0) and100mM TAPS(pH 8.0 -9.5)wereused.

Site-directed mutagenesis. The PCR mega-primermethod was used to produce site-specific mutations in UGP.21 The pET15b-ugp plasmid was used for the site-directed muta

genesis with themutagenicprimers (K194D: 5’- GTG GAA GATCCCGCT CCA-3’; K194R: 5’-GTG GAA AGG CCC GCT -3’),where the underlined text indicatesthe bases where themutation was introduced.

Results and Discussion

The fosmid library of S. chungbukensisDJ77 wasusedto find the ugp gene, whichwas predicted to be inthe cu292 fosmid clone. PCR wasusedtoamplify the full gene fromthe genomicDNA, and the PCRproducts were cloned intothe expressionvector pET15b(pET-15b-ugp). Theinserted gene sequencewas confirmed by DNAsequencing. Overexpression of thegene using an E.coli system wassuccessful, and the6x His-tagged enzyme waspurifiedto homogeneity.Theenzyme displayed one band on SDS-PAGE with Coomassie Blue staining, andthe molecular weight was estimatedtobe approxi mately32kDa,which is consistent with acalculated value of 32.1 kDa(Figure 1).

Amino acid sequence homologyanalysis,usingtranslation databased on the ugp gene sequence of S. chungbukensis DJ77,shows a highdegreeofamino acid sequence homology with the ugp gene of other organisms, particularly S. paucimo- bilis.This homology is likelydueto bothorganismsbelonging to thesamegenus.The UGPfrom the bacteriaZymononasand Novosphingobiumalsoshow high amino acid sequencesimil aritywith that of Sphingomonas. UGPs from other bacteria (e.g., Gluconacetobacter, Rhodopseudomona, Brucella, and Bradyrhizobium)showfairly high similarity (58 - 60% identity) inthe amino acid sequence as well. Table 1 summarizesthe amino acid sequencehomologyanalysis.

A multiple aminoacidsequence alignment of UGP enzymes across several bacteria including S. chungbukensisDJ77 was carriedouttodetermine conservedregions that may play an

Figure 1. SDS-PAGE analysis of UGP. M, molecular markers; Lane 1, crude cell extract from E. coli BL21 (DE3)pLysS without IPTG;

Lane 2, crude cell extract from E. coli BL21 (DE3)pLysS with IPTG;

Lane 3: UGP purified by His-tag purification.

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Soc. , 30, No. 6 Moon-Young al

Table 1. Comparison ofS. chungbukensis DJ77 UGP with other homo

logous proteins.

Organism Product

Identities (%) Positives (%) Sphingomonas paucimobilis

UDP-glucose pyrophosphorylase

217/291 (74) 248/291 (85) Zymomonas mobilis

UTP-glucose-1-phosphate uridyltransferase

183/289 (63) 226/289 (78) Novosphingobium aromaticivorans DSM12444

184/265 (71) 235/292 (80) Gluconacetobacter xylinus

167/284 (58) 215/284 (75) Rhodopseudomona palustris CGA009

UTP-glucose-1-phosphate uridylyltransferase

168/277 (60) 209/277 (75) Brucella suis 1330

166/279 (59) 201/279 (72) Bradyrhizobium japonicum USDA110

167/277 (60) 199/277 (71)

important role inthe UPG enzyme activity. Figure 2 shows theresults of themultiple sequencealignment comparison of UGPenzymes from various organisms. Twoimportant con

servedregionswerefound in the amino acid sequence using theCLUSTALW program (Figure2).The N-terminal region of the UGP containsthe GXGTRXLPATK motif, which is highlyconservedamongst bacterial UGP enzymes.Ithas been suggested that this domain is the activator-binding site.끄23 The other conserved domain amongst bacterial UGPs was foundin theamino acid sequenceshown in Figure 2, which is believed to betheglucose-1-phosphate binding site.22 Wealso

Figure 3. Optimizing the reaction pH for UGP activity. The pH of the assay solution MES (5.5 - 6.5), MOPS (6.5 - 8.0) and TAPS (8.0 - 9.5) buffers.

found a conservedlysineresidue in the binding site, whichis likely toplaya critical role in theenzymaticcatalysis.22

It wasalsoimportant to determine the optimal pH for UGP activity.To do so, we carried out the enzyme assay over a pH range from 5.5 to9.5.Figure3 shows the results of the enzyme activityas a function of pH,indicatingmaximumactivity is obtainedat pH = 8.0.

To determine whether the enzyme has multiple subunits, we used a nativegel, and the resultsareshown in Figure 4. In the denaturinggel (SDS-PAGE),theestimatedmass of the monomer is 32kDa; however, the nativegel displays a band corresponding to anestimatedmass of 63.17kDa(results not shown). This result providesevidencethattheprotein formsahomodimer.

Domain I Domain II

Glucose-l-P-bindingsite Activator-bindingsite

S chungbukensis DJ77 A G L C T R F L P A T K SV P K E L L P (33) L VEKP A P GT APS N (203) S paucimobilis A G L C T R F L P A T K A H PKE N L P (33) L VEKP A P GT APS N (201)

Z. mobilis A G Q C T R F L P A T K AH PKE N L P (31) L VEKP E K -- APS R (200)

G L C T R F L P A T K CV PKE N L TL VEKP DPKD APS T

G. xylinus (32) (202)

A G L C T R V L P A T K AH PKE N L TH VEKP A P GT APS N

R. palustris CGA009 (30) (200)

A G L C T R V L P A T K AH PKE N L TH VEKP A K GT APS N

B. japonicum USDA 110 a

G L C T R F L P A T K S I PKE N L T (30) H

VEKP A K GT APS N (202) B. suis 1330 a G L C T R F L P A T K TI PKE N L T (34) H VEKP K K GT APS N (204) B. Henselae str. a G L C T R F L P A T K AL PKE N L T (33) H VEKP KPKD APS N (204)

N. sp A G L C T R F L P A T K AV PKE N L T (31) H VEKP A K GT APS N (201)

A. tumefaciens str. C58 (34) (204)

*** * **** ***

Figure 2. Multiple alignment of the two characteristic consensus amino acid sequences. The domain I motif contains the activator-binding site of the enzyme; the domain II motif contains a glucose-1-P-binding site of the enzyme. Numbers of intervening amino acid are given in parentheses. S. chungbukensis DJ77, Sphingomonas chungbukensis DJ77; S. paucimobilis, Sphingomonas paucimobilis; Z. mobilis, Zymomonas mobilis; G. xylinus, Gluconecetobacter xylinus; R. palustris CGA009, Rhodopseudomonas palustris CGA009; B. japonicum USDA 110, Bradyrhizobium japonicum USDA 110; B. suis 1330, Brucella suis 1330; B. henselaestr, Bartonella henselae; N. sp, Novosphingobium species; A. tumefaciens str. C58, Agrobacterium tumefaciens str. C58

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Figune 4. Kinetic analysis of UGP. Purified UGP was incubated with varied concentrations of UDP-glucose, and the enzyme activity was determined.

To supportthe hypothesisthat the gene productis UGP, enzyme activity experimentswere carried out via measure

ments designedto monitor theincrease in theabsorbanceat 340nm with increased NADPHconcentration. To examine the effects of substrate concentration onenzymatic activity, theenzyme assay wasconducted over a widerangeof UDP- glucose concentrations. Figure 4 shows the results of the enzymeactivityassayas a functionofUDP-glucoseconcent ration. Theenzymeactivitywashigheratlow substrate con

centration(0 - 0.4 mM) and wasdecreased athigher concent rations of thesubstrate( >0.4 mM),indicatingsubstrate inhibi

tion occursathigher concentrations. Therefore,weconclude thata UDP-glucose concentration of 0.4 mM isoptimal for enzymeactivity.Kinetic data was collected for theconversion ofUDP-glucose to glucose- 1-phosphate. The Kmand Vnax values of the enzymewere measured at pH = 8.0 at a UDP-glucose concentration of0.4 mM, and valuesof1.14 mM for Kmand of

10.09mM/min/mg for Vax weredetermined.

We performed site-directed mutagenesis experiments to determine whether the conserved amino acid, Lys194, is involvedin the enzymeactivity.A charge-reversal mutagenic substitutionwas carried out atLys194by mutation to Asp, and noenzymaticactivity of K194D was detected.This result indicates that Lys194plays a criticalrole in theenzyme me chanism. Inaddition, we also substitutedanArg for Lys194 (which is a conserved substitution of one positive charged sidechain for another). TheKm and V^maxvalues for the Arg- substituted enzyme however, were significantly lowerthan those of thewild-typeenzyme,suggesting that theproximity of theLys residue to the substrate is essential forcatalysis.

Table 2summarizesthe kinetic data for wild-type UPG and its variants.

To date, a Km value of7.5 卩M for S. paucimobilis ATCC 3146111 is the highest value known to theseauthors, and the lowest knownis 3.2 mM forAcetobacterxylimun24Thus, our Km valueof1.14 mM for S. chungbukensis UGP lies within therange for comparablesystems. It shouldbenoted thatthe Km value cannotbe aconclusive value for the affinity between the substrateandthe enzyme, butno informationabout binding

Table 2. Kinetic parameters of the wild type and variant UGPs.

K^m(mM) "^■ax

(mM/min/mg) kcat (s-1) ^kcat/Km (s-1/mM)

Wild-type 1.14 10.09 27.04 23.71

K194D No enzymatic activity

K194R 0.51 1.77 4.74 9.29

constants for the enzyme is available, so we includedtheKm

valuesasaffinityvalues.

We observed substrate inhibition, and to ourknowledge, this kind of inhibition has notbeen reported for any UGP enzymes. The mechanismofthe substrate inhibition was related to a second, non-active sitebinding onthe enzyme, which resultedin a decrease in enzymeactivity at highersubstrate concentrations. In this study weused a coupledmethod in which other moleculesmay act asthe secondsubstrate; however,to identifytheothermolecules,further investigation isneeded.

The Lys194 is located within theVEKP (Val-Glu-Lys-Pro) motif, which is found in many bacterialenzymes,and is predict ed to be essential for substrate binding or catalysis.22 Our site-directed mutagenesis data show that substitution ofthe charge-reversible residue Asp for Lys194 within the consensus sequencemotif dramatically impairs theUGP activity, support ing the hypothesisthat theLys194plays a criticalrole in the catalysis.

In conclusion, it has beendeterminedby gel electrophoresis, amino acidalignment,kineticanalysis, and site-directed muta genesis experiments that our gene product is highlylikelyto be the UGP enzyme. As understandingUGP is essential to EPS biosynthesis,future study should examinethe role ofUGPin the biosynthetic pathway. Thiscouldbedoneby disrupting the genein S. chungbukensis DJ77, and then evaluating the contribu tion of thisenzyme to EPSbiosynthesis in thisbacterium.

Acknowledgments. Thisworkwas supported by theresearch grant from Chungbuk National University in2008. The authors wouldlike to express their appreciation and thankstoProf.

Mark D.Jonklaas atBaylor University for reviewingthis manu script.

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