K-079

Abstract

Background: Escherichia coli has multiple pathways for the salvage of nucleosides. One of these pathways consists of a group of hydrolases capable of breaking down nucleosides to ribose and the corresponding base. E. coli has three different genes for these hydrolases, one of which, rihC, is capable of hydrolyzing both purines and pyrimidines ribonucleosides. Because mammals lack these enzymes, a better understanding of these molecules may make them attractive targets for drug therapy. This study attempted to characterize the active site of the inosine-uridine hydrolase of E. coli, encoded by rihC. Methods: Specific amino acid residues of the rihC gene of E. coli K12 were mutagenized by site-directed mutagenesis using nested polymerase chain reaction. The mutant genes were expressed in a protein expression system and the gene products will be purified and assayed for biological activity of the enzyme. Mathematical analyses will examine essential atoms and dynamics of the reactions that will permit construction of molecular models. Results: Eight clones, each with a different mutation construct in the rihC gene, have been isolated. Protein expression data reveals that the mutant proteins are expressed by each E. coli clone. Measurment of the kinetic activity of mutant proteins are currently in progress. Conclusion: Using the crystal structure of the inosine-uridine hydrolase, a number of amino acids have been identified as potentially important in the interaction of the enzyme with its substrate. Decreased or elimination of enzyme activity with the mutagenized proteins will aid in indentification of the amino acid residues involved in the active site of the enzyme.

Introduction

The death rate reported for malaria, trypanosomiasis, and other infections caused by protozoan parasites exceeds one million per year (1). This number does not address the morbidity of these diseases. This great cost in human life and suffering necessitates the identification of improved treatment for these diseases. A disparity between mammalian and protozoan parasite DNA pathways may provide an adequate target for treatment.Nucleoside hydrolases catalyze the reaction of Nucleoside + H2Oribose + purine or pyrimidine. This reaction is vital for the salvage pathways of protozoan parasites and also is utilized by bacteria. Nucleoside hydrolases, while shown to be vital in the nucleoside salvage pathway of protozoans, are apparently absent in mammals. Most parasitic protozoans lack nucleoside phosporylase which is common in mammals. These differences in the nucleic acid pathways between mammals and protozoans have made the nucleoside hydrolases the target for development of chemotherapeutic agents.

This research focuses on characterizing the active site of a nucleoside hydrolase, rihC, from Escherichia coli. RihC is part of the nucleic acid alternative pathway of E. coli and Salmonella enterica serovar Typhimurium which catalyzes the hydrolysis of different nucleosides to ribose and the corresponding base. Mammals catalyze the release of base by nucleoside phosphorylase. This difference provides the potential for development of antibacterial chemotherapeutic agents.

Methods

Choosing Potential Active Site Amino Acids

•The structure of Inosine-Uridine nucleoside hyrdolase (IUNH) of Crithidia fasciculata has previously been described. The E. coli amino acids to be mutated were chosen based on homology with critical residues of C. fasciculata IUNH.

C.fasc 5 IILDCDPGLDDAVAILLAHGNPEIELLAITTVVGNQTLAKVTRNAQLVADIAGITGVPIA 64 I LD DPG+DDAVAI A PE++L +TTV GN ++ K TRNA + +P+A E.coli 5 IFLDTDPGIDDAVAIAAAIFAPELDLQLMTTVAGNVSVEKTTRNALQLLHFWN-AEIPLA 63 C.fasc 65 AGCDKPLVRKIMTAGHIHGESGMGTVAYPAEFKNKVDERHAVNLIIDLVMSHEPKTITLV 124 G PLVR A +HGESGM + E K A I D +M P+ +TLV E.coli 64 QGAAVPLVRAPRDAASVHGESGMAGYDF-VEHNRKPLGIPAFLAIRDALM-RAPEPVTLV 121 C.fasc 125 PTGGLTNIAMAARLEPRIVDRVKEVVLMGGGYHEGNATSVAEFNIIIDPEAAHIVFNESW 184 G LTNIA+ P ++ +V+MGG GN T AEFNI DPEAA VF E.coli 122 AIGPLTNIALLLSQCPECKPYIRRLVIMGGSAGRGNCTPNAEFNIAADPEAAACVFRSGI 181 C.fasc 185 QVTMVGLDLTHQALATPPILQRVKEVDTNPARFMLEIMDYYTKIYQSNRYMAAAAVHDPC 244 ++ M GLD+T+QA+ TP L + +++ Y + QS M HD C E.coli 182 EIVMCGLDVTNQAILTPDYLSTLPQLNRTGKMLHALFSHYRSGSMQSGLRM-----HDLC 236 C.fasc 245 AVAYVIDPSVMTTERVPVDIELTGKLTLGMTVADFRNPRPEHCHTQVAVKLDFEKFWGLV 304 A+A+++ P + T + V +E G+ T G TV D + + QVA+ LD + F V E.coli 237 AIAWLVRPDLFTLKPCFVAVETQGEFTSGTTVVDIDGCLGKPANVQVALDLDVKGFQQWV 296 C.fasc 305 LDAL 308 + L E.coli 297 AEVL 300

Figure 2. Crithidia fasciculata has a well-characterized inosine-uridine hydrolase. The active site of C. fasciculata is known. Homology between IUNH of C. fasciculata and the E. coli rihC gene product for the choice of amino acid residues to be mutated (shown in yellow). Alignment was performed with BlastP.

Pathways of Pyrimidine and Purine Metabolism in E.coli

Figure 1. Pathways of pyrimidine and purine metabolism in E. coli. rihC is shown to hydrolyze multiple substrates and is the only hydrolase shown to act on purines

Mutagenesis and Expression of the Inosine-Uridine Hydrolase Gene from Escherichia coli

Brock Arivett1, Mary Farone1, Paul Kline2, Terrance Quinn3, Abdul Khaliq1, Zachariah Sinkala3, and Anthony Farone1

*Department of Biology1, Chemistry2, and Mathematics3

Middle Tennessee State University, Murfreesboro, Tennessee

Plasmid Construct and Expression

•rihC was inserted between Nco I and Xho I of pET-28b restriction sites (Novagen)

Site-Directed Mutagenesis and DNA Sequencing

• Stratagene QuiKChange® Site-Directed Mutagenesis Kit was used to produce the desired mutations

Site-Directed Mutagenesis Primers Primer Name Primer Sequence (5` to 3`)

D14A 5`-ACCCCGGCATTGCCGATGCCGTCGC-3` D14A antisense 5`-GCGACGGCATCGGCAATGCCGGGGT-3` D15A 5`-CCCGGCATTGACGCTGCCGTCGCCATT-3` D15A antisense 5`-AATGGCGACGGCAGCGTCAATGCCGGG-3` F164A 5`-TGTACGCCAAACGCCGAGGCTAATATTGCTGCCATCC-3` F164A antisense 5`-GGATCGGCAGCAATATTAGCCTCGGCGTTTGCGTACA-3` R222A 5`-CCCTGTTTAGCCACTACGCTAGCGGCAGTATGCAAA-3` R222A antisense 5`-TTTGCATACTGCCGCTAGCGTAGTGGCTAAACAGGG-3` H233A 5`-GCGGCTTGCGAATGGCCGATCTCTGCGCCA-3` H233A antisense 5`-TGGCGCAGAGATCGGCCATTCGCAAGCCGC-3` D234A 5`-CTTGCGAATGCACGCTCTCTGCGCCATCG-3` D234A antisense 5`-CGATGGCGCAGAGAGCGTGCATTCGCAAG-3` L241A 5`-GCCATCGCCTGGGCGGTGCGCCCGGA-3` L241A antisense 5`-TCCGGGCGCACCGCCCAGGCGATGGC-3` V242A 5`-CGCCTGGCTGGCGCGCCCGGACC-3` V242A antisense 5`-GGTCCGGGCGCGCCAGCCAGGCG-3`

rihC Amino Acid sequence 0 MRLPIFLDTD PGIDDAVAIA AAIFAPELDL QLMTTVAGNV SVEKTTRNAL QLLHFWNAEI 61 PLAQGAAVPL VRAPRDAASV HGESGMAGYD FVEHNRKPLG IPAFLAIRDA LMRAPEPVTL 121 VAIGPLTNIA LLLSQCPECK PYIRRLVIMG GSAGRGNCTP NAEFNIAADP EAAACVFRSG 181 IEIVMCGLDV TNQAILTPDY LSTLPQLNRT GKMLHALFSH YRSGSMQSGL RMHDLCAIAW 241 LVRPDLFTLK PCFVAVETQG EFTSGTTVVD IDGCLGKPAN VQVALDLDVK GFQQWVAEVL 301 ALAS

Figure 3. Diagram of pET-28b plasmid

Protein Modeling

• Visual Molecular Dynamics Software was utilized to prepare likely structural images.

Substrate Screening

•Multiple nucleosides were screened using UV-HPLC to determine appropriate substrates of wild-type rihC product.

N

NN

N

NH2

O

HOH

OHH

HH

HO

Arabino adenosine

N

NN

N

Cl

O

OHOH

HH

HH

HO

6-chloropuine riboside

NH

O

ON

O

OHOH

HH

H

H

H

Erythrouridine

N

NH2

ON

O

OHOH

HH

HH

HO

cytidine

NH

O

ON

O

OHOH

HH

HH

HO

Uridine

N

NN

N

NH2

O

OHOH

HH

HH

HO

Adenosine

NH

NN

N

O

OHOH

HH

HH

HO

O

Inosine

NH

NH

N

N

O

O

OHOH

HH

HH

HO

O

xanthosine guanosine

NH

N

N

O

NH2N

O

OHOH

HH

HH

HO

O

HOH

HH

HH

HO

N

N

NH2

O

deoxycitidine

N

NN

N

NH2

O

HOH

HH

HH

HO

Deoxyadenosine

Mutant Protein Expression, Purification, and Activity Analysis

• The production of purified mutant protein will be performed on a metal affinity column. This takes advantage of the 6 histidine residues designed into the amino acid sequence.

Figure 4. Nucleosides screened as supbstrate of rihC

Table 1. Site-Directed Mutagenesis primers used to produce amino acid changes. The specific amino acid and primer are color coded.

•The protein is expressed in E. coli BL21 (DE3) pLysS cells (Novagen)

Contact Information:

Mary B. Farone, Ph.D.Biology Department

Middle Tennessee State UniversityMurfreesboro, TN 37132

mfarone@mtsu.edu

Conclusions

• Based upon the results of the sequence data the desired mutation were encoded via Site-Directed Mutagenesis.

• The VMD models show the amino acids of interest being located in a shared region of the enzyme.

• The substrate screening indicates rihC having the ability to catalyze the hydrolysis of many nucleosides.

• The substrate screening also indicates the enzyme activity is greatly inhibited by the any change in the ribose of the nucleosides.

Literature Cited

Hunt C, Gillani N, Farone A, Rezaei M, Kline PC. 2005. Kinetic isotope effects Hunt C, Gillani N, Farone A, Rezaei M, Kline PC. 2005. Kinetic isotope effects of nucleoside hydrolase from of nucleoside hydrolase from Escherichia coli.Escherichia coli. Biochimica et Biophysica Acta. Biochimica et Biophysica Acta. 1251:140149.1251:140149.

Giabbai B, Degano M. 2004. Crystal structure to 1.7 angstrom of the Giabbai B, Degano M. 2004. Crystal structure to 1.7 angstrom of the Escherichia coliEscherichia coli pyrimidine nucleoside hydrolase yeiK, a novel candidate for cancer gene therapy. pyrimidine nucleoside hydrolase yeiK, a novel candidate for cancer gene therapy. Structure. 12:739-749.Structure. 12:739-749.

Petersen C, Moller LB. 2001. The rihA, rihB, and rihC ribonucleoside hydrolases of Petersen C, Moller LB. 2001. The rihA, rihB, and rihC ribonucleoside hydrolases of Escherichia coliEscherichia coli substrate specificity,gene expression, and regulation. J. Biol. substrate specificity,gene expression, and regulation. J. Biol. Chem. 276:884-894.Chem. 276:884-894.

Horenstein BA, Parkin DW, Estupinan B, Schramm VL. 1991. Transition-state Horenstein BA, Parkin DW, Estupinan B, Schramm VL. 1991. Transition-state analysis of nucleoside hydrolase from analysis of nucleoside hydrolase from Crithidia fasciculataCrithidia fasciculata. Biochemistry. . Biochemistry. 30:10788-10795.30:10788-10795.

Gopaul DN, Myer SL, Degano M, Sacchettini JC, Schramm VL. 1996. Inosine-uridine Gopaul DN, Myer SL, Degano M, Sacchettini JC, Schramm VL. 1996. Inosine-uridine nucleoside hydrolase from nucleoside hydrolase from Crithidia fasciculata Crithidia fasciculata genetic characterization, genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue. crystallization, and identification of histidine 241 as a catalytic site residue. Biochemistry. 35:5963-5870.Biochemistry. 35:5963-5870.

Acknowledgements

The wildtype plasmid construct was generously provided by Dr. Massimo Degano. GenHunter of Nashvile, TN provided sequence data. This work was funded in part by continuing support from the Office of Graduate Studies Research Enhancement Program, Biology and Chemistry departments of MTSU.

Results

DNA Sequences

• Sequences acquired from GenHunter Corporation (Nashville, TN) confirmed all single amino acid mutants were obtained.

Protein Models

Figure 6. (A) Wildtype rihC protein. (B) Location of all mutated amino acid residues.

Substrate Screening

Protein Expression and Purification

Wildtype rihC Nucleoside Substrate Screening

Nucleoside Hydrolysis

Arabino adenosine No 6-chloropurine riboside

Yes

Erythrouridine No Cytidine Yes Uridine Yes Adensosine Yes Inosine Yes Xanthosine Yes Guanosine Yes Deoxycytidine No Deoxyadenosine No

Table 2. Nucleosides tested as potential substrates of wild-type rihC.

A B

Figure 5. Coomassie staining of His-select purification of wildtype rihC.