* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Pathways of Pyrimidine and Purine Metabolism in E.coli
Biochemical cascade wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Peptide synthesis wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Western blot wikipedia , lookup
Gene regulatory network wikipedia , lookup
Gene expression wikipedia , lookup
Magnesium transporter wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Catalytic triad wikipedia , lookup
Proteolysis wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Genetic code wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Metalloprotein wikipedia , lookup
Point mutation wikipedia , lookup
Protein structure prediction wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Expression vector wikipedia , lookup
Biochemistry wikipedia , lookup
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. 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 Methods Choosing Potential Active Site Amino Acids 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. •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 E.coli 5 C.fasc 65 E.coli 64 IILDCDPGLDDAVAILLAHGNPEIELLAITTVVGNQTLAKVTRNAQLVADIAGITGVPIA 64 I LD DPG+DDAVAI A PE++L +TTV GN ++ K TRNA + +P+A IFLDTDPGIDDAVAIAAAIFAPELDLQLMTTVAGNVSVEKTTRNALQLLHFWN-AEIPLA 63 AGCDKPLVRKIMTAGHIHGESGMGTVAYPAEFKNKVDERHAVNLIIDLVMSHEPKTITLV 124 G PLVR A +HGESGM + E K A I D +M P+ +TLV 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. Mutagenesis and Expression of the InosineUridine Hydrolase Gene from Escherichia coli 1 Brock Arivett , 1 Farone , Mary Paul Terrance 3 1 Zachariah Sinkala , and Anthony Farone 1 Biology , 2 Kline , 3 Quinn , Abdul 2 Chemistry , 3 Mathematics 1 Khaliq , *Department of and Middle Tennessee State University, Murfreesboro, Tennessee Plasmid Construct and Expression Protein Modeling •rihC was inserted between Nco I and Xho I of pET-28b restriction sites (Novagen) • 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. NH2 N NH2 N N Cl N N N N N O HO Arabino adenosine H O O OH H H H cytidine H OH N HO HO O N H H H OH OH H H OH 6-chloropuine riboside H H OH O O NH2 NH N NH N N Figure 3. Diagram of pET-28b plasmid N H •The protein is expressed in E. coli BL21 (DE3) pLysS cells (Novagen) O H N HO O H OH OH H H OH Adenosine O H Uridine H H N HO Erythrouridine O O H H H OH OH H H OH O O O N N NH NH N NH N Site-Directed Mutagenesis and DNA Sequencing N HO N HO N O N H HO O O H O H Inosine H H OH H H H xanthosine OH OH NH2 N N deoxycitidine MRLPIFLDTD PLAQGAAVPL VAIGPLTNIA IEIVMCGLDV LVRPDLFTLK ALAS PGIDDAVAIA VRAPRDAASV LLLSQCPECK TNQAILTPDY PCFVAVETQG AAIFAPELDL HGESGMAGYD PYIRRLVIMG LSTLPQLNRT EFTSGTTVVD QLMTTVAGNV FVEHNRKPLG GSAGRGNCTP GKMLHALFSH IDGCLGKPAN SVEKTTRNAL IPAFLAIRDA NAEFNIAADP YRSGSMQSGL VQVALDLDVK QLLHFWNAEI LMRAPEPVTL EAAACVFRSG RMHDLCAIAW GFQQWVAEVL O H OH H N HO H H N O HO H 0 61 121 181 241 301 OH N O Primer Name D14A D14A antisense D15A D15A antisense F164A F164A antisense R222A R222A antisense H233A H233A antisense D234A D234A antisense L241A L241A antisense V242A V242A antisense guanosine NH2 N Site-Directed Mutagenesis Primers Primer Sequence (5` to 3`) 5`-ACCCCGGCATTGCCGATGCCGTCGC-3` 5`-GCGACGGCATCGGCAATGCCGGGGT-3` 5`-CCCGGCATTGACGCTGCCGTCGCCATT-3` 5`-AATGGCGACGGCAGCGTCAATGCCGGG-3` 5`-TGTACGCCAAACGCCGAGGCTAATATTGCTGCCATCC-3` 5`-GGATCGGCAGCAATATTAGCCTCGGCGTTTGCGTACA-3` 5`-CCCTGTTTAGCCACTACGCTAGCGGCAGTATGCAAA-3` 5`-TTTGCATACTGCCGCTAGCGTAGTGGCTAAACAGGG-3` 5`-GCGGCTTGCGAATGGCCGATCTCTGCGCCA-3` 5`-TGGCGCAGAGATCGGCCATTCGCAAGCCGC-3` 5`-CTTGCGAATGCACGCTCTCTGCGCCATCG-3` 5`-CGATGGCGCAGAGAGCGTGCATTCGCAAG-3` 5`-GCCATCGCCTGGGCGGTGCGCCCGGA-3` 5`-TCCGGGCGCACCGCCCAGGCGATGGC-3` 5`-CGCCTGGCTGGCGCGCCCGGACC-3` 5`-GGTCCGGGCGCGCCAGCCAGGCG-3` rihC Amino Acid sequence H OH • Stratagene QuiKChange® Site-Directed Mutagenesis Kit was used to produce the desired mutations Table 1. Site-Directed Mutagenesis primers used to produce amino acid changes. The specific amino acid and primer are color coded. H H H OH H N Deoxyadenosine H H OH H H H Figure 4. Nucleosides screened as supbstrate of rihC 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. NH2 Contact Information: Mary B. Farone, Ph.D. Biology Department Middle Tennessee State University Murfreesboro, TN 37132 [email protected] Results DNA Sequences • Sequences acquired from GenHunter Corporation (Nashville, TN) confirmed all single amino acid mutants were obtained. Protein Expression and Purification 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. Figure 5. Coomassie staining of His-select purification of wildtype rihC. Protein Models B A Literature Cited Hunt C, Gillani N, Farone A, Rezaei M, Kline PC. 2005. Kinetic isotope effects of nucleoside hydrolase from Escherichia coli. Biochimica et Biophysica Acta. 1251:140149. Giabbai B, Degano M. 2004. Crystal structure to 1.7 angstrom of the Escherichia coli pyrimidine nucleoside hydrolase yeiK, a novel candidate for cancer gene therapy. Structure. 12:739-749. Petersen C, Moller LB. 2001. The rihA, rihB, and rihC ribonucleoside hydrolases of Escherichia coli substrate specificity,gene expression, and regulation. J. Biol. Chem. 276:884-894. Horenstein BA, Parkin DW, Estupinan B, Schramm VL. 1991. Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata. Biochemistry. 30:10788-10795. Figure 6. (A) Wildtype rihC protein. (B) Location of all mutated amino acid residues. Substrate Screening Gopaul DN, Myer SL, Degano M, Sacchettini JC, Schramm VL. 1996. Inosine-uridine nucleoside hydrolase from Crithidia fasciculata genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue. Biochemistry. 35:5963-5870. Acknowledgements Table 2. Nucleosides tested as potential substrates of wild-type rihC. Wildtype rihC Nucleoside Substrate Screening Nucleoside Arabino adenosine 6-chloropurine riboside Erythrouridine Cytidine Uridine Adensosine Inosine Xanthosine Guanosine Deoxycytidine Deoxyadenosine Hydrolysis No Yes No Yes Yes Yes Yes Yes Yes No No 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.