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ISSN 2348-6775 (Online)
International Journal of Interdisciplinary Research
2349-5480 (Print)
HOMOLOGY MODELING OF PHOSPHOMETHYL PYRIMIDINE
KINASE FROM LEPTOSPIRA INTERROGANS
Farooqe E Azam
University Institute of Engineering and Technology, Chattrapati Shahu Ji
Maharaj University, Kanpur – 208 024
Shilpa Kaistha
University Institute of Engineering and Technology, Chattrapati Shahu Ji
Maharaj University, Kanpur – 208 024
ABSTRACT
Leptospira interrogans causes the leptospirosis disease. Treatment for severe leptospiral disease is still
unclear. Therefore, it is important to design effective antileptospiral drugs. A molecular model of the
phosphomethyl pyrimidine kinase from leptospira interrogans is documented in this study. The predicted
model is believed to provide valuable insights towards inhibitor design of phosphomethyl pyrimidine kinase
pertaining to leptospirosis treatment.
Key words- Leptospira interrogans, phosphomethyl pyrimidine kinase, homology modeling.
Introduction
Leptospira is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species.
The first leptospira to be described was L. interrogans in 1907(as Spirochaeta interrogans) was isolated from
kidney tissue slices of a leptospirosis victim.
Leptospira interrogans is an important mammalian pathogen. Bacteria were grown for several days after
culture temperatures were shifted from 300 to 370 C. Triton X-114 cellular fractionation identified several
proteins of the cytoplasm, periplasm and outer membrane for which synthesis was dependent on the culture
temperature.
Genome Structure
L. interrogans serogroup Icterhaemorrhagiae consists of a 4.33 megabase large chromosome and a 359
kilobase small chromosome, totaling 4,768 predicted genes. A series of genes have been discovered that could
potentially be related to adhesion. This genome differs from the two other pathogenic spirochaetes
(Treponema pallidum and Borrelia burgdorferi), though some similar genes are visible.
Cell structure and Metabolism
Leptospira cells elongate as they grow, closing off the fresh ends. Sometimes when under nutritional stress,
leptospiires over 50µm long may fail to separate and create elongated chains of leptospires. Leptospira
organisms are chromatographs, using O2 as the electron receptor. Long chain fatty acids are the sole major
enrgy source, derived through β-oxidation, though they cannot be synthesized through the tri carboxylic acid
cycle(B.Adler et al., 2004).
Ecology
Leptospira is a unique pathogen because of its ability to grow at temperatures as low as 11-130 C though the
optimum growth temperature is still between 7.2 and 7.6, preferring an alkaline habitat to acidic(B. Adler et al.,
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2004). Leptospirosis, caused by leptospira, is found most frequently in tropical or temperate climates (CDC,
2003).
Pathology
Leptospira interrogans causes the disease Leptospirosis:
Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of
morbidity and mortality in highly populated tropical urban centres. Leptospira are flexible, gram negative
spirochaetes (cork-screw or spiral shaped bacteria) that are six to nine micro meters long. These microbes
propel themselves using internal flagella and twisting back and forth.
Leptospira is a spiral shaped spirochaete –
Leptospira cause leptospirosis and although leptospirosis has long been recognized as an important endemic
disease in many tropical regions, it is now becoming a more common problem in highly populated urban
centres. Clinically it is typified by diverse symptoms that include fever, headache, severe muscle pain, ocular
disorders, meningitis, jaundice, renal failure and pulmonary haemorrhage.
Transmission is most often from dogs, livestock, and wild mammals. The organisms can establish a commensal
relationship with many animal hosts, persisting in the renal tubules without producing disease or causing
pathologic changes in the kidney. From here they are continually excreted in the urine and can contaminate
natural bodies of water which serve as a source of the infection. The organism penetrates intact mucous
membranes or burrows through small breaks in the skin to enter the blood stream although occasionally it is
transmitted through ingestion of contaminated food or by inhalation of droplets aerosols of contaminated
fluids.
Leptospira are unique invasive spirochaetes in that they contain outer membrane lipopolysaccharide (LPS) as
well as lipoprotein.
Leptospirosis can cause jaundice. From Michaele Lowe
Leptospirosis, a potentially deadly disease, is caused by the spirochaete leptospira. Leptospirosis affects both
humans and animals, causing a wide range of symptoms in both. In humans, typical symptoms can include
fever, headache, chills, sore muscles, vomiting, jaundice, red eyes, abdominal pain, diarrhoea and rashes.
Leptospirosis can become considerably dangerous if not treated, potentially leading to kidney damage,
meningitis, liver failure and respiratory problems. Leptospirosis is typically contracted by humans through
water, food, or urine contact with an infected humans contract. Leptospirosis through contact with dogs and
about one third from contact with rats. (Mar Vista Vet, 2004).
Leptospirosis affects animals severely as well. Once in contact with the disease, the organism spread through
the bloodstream then settle in the kidneys to reproduce. In dogs, this leads to fever, loss of appetite, joint pain,
nausea, excessive drinking and jaundice. Organ failure can occur as well, especially in the liver (Mar Vista Vet,
2004).
Diseases
Anicteric leptospirosis (Fort Bragg Fever)
Fever, chills, headache, myalgia, muscle pain, eye pain, photophobia, conjunctivitis, abdominal pain, nausea,
vomiting, lymphadenopathy, splenomegaly, rash, meningeal symptoms.
Icteric leptospirosis (weil’s syndrome)
Fever, chills, headache, myalgia, muscle pain, cough, enteric fever syndrome, abdominal pain, nausea,
vomiting, diarrhoea, uremia, jaundice, hepatomegaly, hematuria.
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Aseptic meningitis
Fever, headache, sore throat, nausea, vomiting, stiff neck, nuchal rigidity, acute confusion, paresis, transient
paralysis.
Methodology
The protein sequence (214 residues long) was obtained from the SWISS PROT database. The protein databank
(PDB) contains two phosphomethyl pyrimidine kinase structures from 2 species (Salmonella typhimurium (PDB
ID: 1JXH), Thermus thermophilus HB8 (PDB ID: 1UB0)). However, structure for phosphomethyl pyrimidine
kinase from Leptospira interrogans was not available. Sequence analysis using BLASTP against PDB sequences
showed phosphomethyl pyrimidine kinase from Leptospira interrogans having 34.3% sequence identity
(highest homology compared to other known structures) with Thermus thermophilus HB8. Therefore we used
the structure of Thermus thermophilus HB8 phosphomethyl pyrimidine kinase (PDB: 1UB0) as template for
building homology model for Leptospira interrogans phosphomethyl pyrimidine kinase using Geno 3D by
satisfaction of spatial restraints. MALIGN script of Geno 3D was implemented to build global alignment in this
modeling process. The obtained alignment is shown in Figure.
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Results and discussion
An active phosphomethyl pyrimidine kinase is a 214 residue long monomer with a molecular mass of 29 kDa. A
similar result has been documented earlier in E. coli ThiE protein. The protein molecule is an alpha beta
structure identified to contain a single large domain (17-214 residues) only one ATP binding motif was found
in its ThiE. It is a two step mechanism for phosphorylation is still unclear. In the current work, only a single
large domain has been identified.
Possibly the phosphorylation process in leptospira species also follows the not so well understood two step
mechanism. Walker et al (1984) suggested that many ATP- and GTP-binding proteins contain a consensus
sequence motif [AG]X%GK[ST]. A sequence similar to this motif, GGGAGIQADLKT, is found in the ThiE
sequence starting at position 19 from the N terminus. Previously a similar sequence motif was also reported in
E. coli. This indicates the domain identified in phosphomethyl pyrimidine kinase could be an ATP binding
motif.
Conclusion
Deoxycycline or penicillin drugs are commonly used for treatment of mild or subclinical leptospiral infections.
Treatment for severe leptospiral disease is still unclear. Therefore, it is important to design effective
antileptospiral drugs. A molecular model of the phosphomethyl pyrimidine kinase from leptospira interrogans
is documented in this study. The predicted model is believed to provide valuable insights towards the inhibitor
design of phosphomethyl pyrimidine kinase pertaining to leptospirosis treatment.
References
1.
Adler, B., S. Faine. “The Prokaryotes: An Evolving Electronic Resource for the Microbiological
Community”. 2004. Springer-Verlag new York, LLC.
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A. Sali & T.L. Blundell, J. Mol. Biol., 234:779(1993) [PMID:8254673].
Centers for Disease Control and Prevention. Division of Bacterial and Mycotic Diseases 7 Mar 2003.
Chinese National Human Genome Centre at Shanghai (CHGC). 2004. Unique physiological and
pathogenic features of Leptospira interrogans revealed by whole genome sequencing.
http://www.rcsb.org/pdb
G. N. Ramachandran et al., J. Mol. Biol., 7:95(1963) [PMID:13990617]
http://bioinfo.hku.hk/Leptolist/
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http://www.nature.com/cgitaf/DynaPage.taf?file=/ni/journal/v2/n4/ab s/ni0401_346_fs.html
http://genomenewsnetwork.org/sequenced_genomes/genome_guide_p2.shtml#lepto
J.E. Walker et al., J.Biochem. Biophys. Acta., 768:164 (1984)
J. Bacteriol. 186(7):2164-2172(2004)
Levett PN, Morey RE, Steigerwalt AG, Ellis WA. “Reclassification of Leptospira parva Hovind-Hougen et
al. 1982 as Turneriella parva gen. nov., comb. Nov.” Int J Syst Evol Microbiol. 2005 Jul; 55(Pt 4):14971499.
L. Parini. 2004. II Libro Scientifico.
Mar Vista Vet.2004. Canine Leptospirosis.
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