K079

1
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
• 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.

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.
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.
2
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)

• 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.

6-chloropuine riboside
cytidine
Figure 3. Diagram of pET-28b plasmid
Erythrouridine
Adenosine
Uridine

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

• Site-Directed Mutagenesis and DNA Sequencing
• Stratagene QuiKChange Site-Directed Mutagenesis
  Kit was used to produce the desired mutations

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

3
Contact Information Mary B. Farone,
Ph.D. Biology Department Middle Tennessee State
University Murfreesboro, TN 37132 mfarone_at_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.

Results

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

Protein Expression and Purification
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. 1251140149. 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. 12739-749. Petersen C,
Moller LB. 2001. The rihA, rihB, and rihC
ribonucleoside hydrolases of Escherichia coli
substrate specificity,gene expression, and
regulation. J. Biol. Chem. 276884-894. Horenst
ein BA, Parkin DW, Estupinan B, Schramm VL.
1991. Transition-state analysis of nucleoside
hydrolase from Crithidia fasciculata.
Biochemistry. 3010788-10795. 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.
355963-5870.
Figure 5. Coomassie staining of His-select
purification of wildtype rihC.
Protein Models
B
A
Figure 6. (A) Wildtype rihC protein. (B)
Location of all mutated amino acid residues.
Substrate Screening
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.
Table 2. Nucleosides tested as potential
substrates of wild-type rihC.