Transcript Pathways of Pyrimidine and Purine Metabolism in E.coli

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 +
H2Oribose + 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.