TargetSp15+-+Isoleucine-tRNA+Synthetase,+Putative+(Toxoplasma+Gondii)


 * // Toxoplasma gondii //**


 * Target (protein/gene name): ** Isoleucine-tRNA synthetase, putative


 * NCBI Gene # or RefSeq#: ** 7896257


 * Protein ID (NP or XP #) or Wolbachia#: ** XP_002369568.1


 * Organism (including strain): ** //Toxoplasma gondii// (strain: ME49)


 * Etiologic Risk Group (see link below): ** Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents

Toxoplasmosis is a resourceful and global infection that results from the intracellular protozoan parasite //Toxoplasma gondii.// The disease was discovered by scientists researching in North Africa and Brazil around 100 years ago, and was first documented in a 3-day-old infant from the New York area in 1934. The severe pathogenic capabilities of //Toxoplasma gondii// were truly recognized in the 1920s and 1930s, namely in congenitally infected children experiencing a few key symptoms such as hydrocephalus, retinochoroiditis and encephalitis; it was also linked to severe ocular inflammation. It was not until the 1980s, however, when //Toxoplasma gondii// emerged as a major cause of death in patients—this further illustrated the public health concern in finding a control for the infection. Additionally, researchers first began questioning the potential pathways of the //Toxoplasma gondii// parasite when it started being reported as an essential factor in New Zealand sheep abortions in the 1950s. Later in the 1960s, an even more essential discovery was made that furthered the research of Toxoplasmosis: cats were the definitive hosts. This finding aided in scientists’ understanding of //Toxoplasma gondii’s// life cycle, and how the “oocyst stage of [the parasite that was] shed in the faeces of infected cats was found to be an important source of” the disease; the stage is apparently very vigorous and can potentially survive in an environment for up to 12-18 months. This also gave an explanation of why the infection was found in herbivorous animals and individuals who are vegetarians. Toxoplasmosis is a global disease that can essentially infect all warm-blooded hosts, and due to its high prevalence rate in certain areas of the world, it continues to pose a significant health concern.
 * Disease Information: **


 * Link to TDR Targets page: ** []


 * Link to Gene Database page: ** []

[] []
 * Essentiality of this protein: ** Very essential in the biosynthesis and metabolism of proteins


 * Is it a monomer or multimer as biological unit? ** (make prediction at [] ): Monomer (1 amino acid chain and 2 ligands in ASU)


 * Complex of proteins?: ** Monomer in solutions

[] [] [] ?
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **


 * EC#: ** 6.1.1.5


 * Link to BRENDA EC# page: ** []

Figure 1. Mechanism of Isoleucine-tRNA synthetase, putative in enzyme assay.


 * Enzyme Assay information: ** “Reactions were performed in a 50μl volume with rIleRS enzyme (40 μg/ml), L-isoleucine (0.1, 0.5, 1, 5, 10 and 50 mM), 8 mu;M of tRNAIle, 2u/ml PPiase, 100μM ATP, and 1mM DTT in aminoacylation buffer. Reactions were incubated at 37°C; at 2.5, 5 and 10 minutes, 15 μl aliquots were withdrawn and mixed with 10 mM EDTA on ice to stop the reactions. 100 μl of malachite green solution was added, and the absorbance measured at 620 nm.”


 * Link to Sigma (or other company) page for assay ** : Told not to do

[]
 * Link (or citation) to paper that contains assay information: **

ATP + L-isoleucine + tRNAIle = AMP + diphosphate + L-isoleucyl-tRNAIle
 * Links to assay reagents (substrates): **

vial of ~1 mg ATP (Sigma), FLAAS-5VL: $120.00 L-isoleucine reagent grade, >98% (HPLC) (Sigma-Aldrich), I2752-100G: $127.00 lyophilized powder (Sigma ), R6018-1UN: $42.00
 * List cost and quantity of substrate reagents, supplier, and catalog #: **

[]
 * Current Inhibitors: ** “Mupirocin, currently in clinical use for the topical treatment of //Staphylococcus aureus//, that acts through the inhibition of the isoleucyl-tRNA synthetase”


 * Expression Information (has it been expressed in bacterial cells): ** Expressed in E. coli cells


 * Purification Method: ** Recombinant His-tagged wild-type and mutants enzymes from Escherichia coli by nickel affinity chromatography

Figure 2. PDB protein structure of Isoleucine-tRNA synthetase, putative.
 * Structure (PDB or Homology model): 1ILE **

RAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACRESVFTYEKEWEAFTERIAYWVDLEDAYATLEPTYIESIWW SLKNLFDRGLLYRDHKVVPYCPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTQVLKTFPGKALEGLPYTPPYPQALEKGYFVVLADYVSQEDGTGIVHQ APAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFREANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYA TESWFIKNTLFKDELIRNNQEIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFRESFPADFIAEGIDQTRGWFN SLHQLGVMLFGSIAFKNVICHGLILDEKGQKMSKSKGNVVDPWDIIRKFGADALRWYIYVSAPPEADRRFGPNLVRETVR DYFLTLWNVYSFFVTYANLDRPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRLEAKESVHLADWPEADPALADEALVA QMRAVLKVVDLARAARAKSGV
 * Amino Acid Sequence: ** MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQARSYKDLFPRYKTMRGYYAPR


 * Molecular Weight: ** 94813.56 kD


 * Length: ** 821


 * Extinction coefficients: **

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 200705 Abs 0.1% (=1 g/l) 2.123, assuming all pairs of Cys residues form cystines

Ext. coefficient 200080 Abs 0.1% (=1 g/l) 2.116, assuming all Cys residues are reduced

Figure 3. TMpred graph of Isoleucine-tRNA synthetase, putative.
 * TMpred graph Image ** :

atgtttaaagaagtgggcgaaccgaactttccgaaactggaagaagaagtgctggcgttt tggaaacgcgaaaaaatttttcagaaaagcgtggaaaaccgcaaaggcggcccgcgctat accgtgtatgaaggcccgccgaccgcgaacggcctgccgcatgtgggccatgcgcaggcg cgcagctataaagatctgtttccgcgctataaaaccatgcgcggctattatgcgccgcgc cgcgcgggctgggatacccatggcctgccggtggaactggaagtggaaaaaaaactgggc ctgaaaagcaaacgcgaaattgaagcgtatggcattgaacgctttaaccaggcgtgccgc gaaagcgtgtttacctatgaaaaagaatgggaagcgtttaccgaacgcattgcgtattgg gtggatctggaagatgcgtatgcgaccctggaaccgacctatattgaaagcatttggtgg agcctgaaaaacctgtttgatcgcggcctgctgtatcgcgatcataaagtggtgccgtat tgcccgcgctgcggcaccccgctgagcagccatgaagtggcgctgggctataaagaaatt caggatccgagcgtgtatgtgcgctttccgctgaaagaaccgaaaaaactgggcctggaa aaagcgagcctgctgatttggaccaccaccccgtggaccctgccgggcaacgtggcggcg gcggtgcatccggaatatacctatgcggcgtttcaggtgggcgatgaagcgctgattctg gaagaaggcctgggccgcaaactgctgggcgaaggcacccaggtgctgaaaacctttccg ggcaaagcgctggaaggcctgccgtataccccgccgtatccgcaggcgctggaaaaaggc tattttgtggtgctggcggattatgtgagccaggaagatggcaccggcattgtgcatcag gcgccggcgtttggcgcggaagatctggaaaccgcgcgcgtgtatggcctgccgctgctg aaaaccgtggatgaagaaggcaaactgctggtggaaccgtttaaaggcctgtattttcgc gaagcgaaccgcgcgattctgcgcgatctgcgcggccgcggcctgctgtttaaagaagaa agctatctgcatagctatccgcattgctggcgctgcagcaccccgctgatgtattatgcg accgaaagctggtttattaaaaacaccctgtttaaagatgaactgattcgcaacaaccag gaaattcattgggtgccgccgcatattaaagaaggccgctatggcgaatggctgaaaaac ctggtggattgggcgctgagccgcaaccgctattggggcaccccgctgccgatttgggtg tgccaggcgtgcggcaaagaagaagcgattggcagctttcaggaactgaaagcgcgcgcg accaaaccgctgccggaaccgtttgatccgcatcgcccgtatgtggatcaggtggaactg gcgtgcgcgtgcggcggcaccatgcgccgcgtgccgtatgtgattgatgtgtggtatgat agcggcgcgatgccgtttgcgagcctgcattatccgtttgaacatgaagaagtgtttcgc gaaagctttccggcggattttattgcggaaggcattgatcagacccgcggctggtttaac agcctgcatcagctgggcgtgatgctgtttggcagcattgcgtttaaaaacgtgatttgc catggcctgattctggatgaaaaaggccagaaaatgagcaaaagcaaaggcaacgtggtg gatccgtgggatattattcgcaaatttggcgcggatgcgctgcgctggtatatttatgtg agcgcgccgccggaagcggatcgccgctttggcccgaacctggtgcgcgaaaccgtgcgc gattattttctgaccctgtggaacgtgtatagcttttttgtgacctatgcgaacctggat cgcccggatctgaaaaacccgccgccgccggaaaaacgcccggaaatggatcgctggctg ctggcgcgcatgcaggatctgattcagcgcgtgaccgaagcgctggaagcgtatgatccg accaccagcgcgcgcgcgctgcgcgattttgtggtggaagatctgagccagtggtatgtg cgccgcaaccgccgccgcttttggaaaaacgaagatgcgctggatcgcgaagcggcgtat gcgaccctgtatgaagcgctggtgctggtggcgaccctggcggcgccgtttaccccgttt ctggcggaagtgctgtggcagaacctggtgcgcagcgtgcgcctggaagcgaaagaaagc gtgcatctggcggattggccggaagcggatccggcgctggcggatgaagcgctggtggcg cagatgcgcgcggtgctgaaagtggtggatctggcgcgcgcggcgcgcgcgaaaagcggc gtg
 * CDS Gene Sequence: **


 * GC% Content for gene: ** 57.2473


 * CDS Gene Sequence (codon optimized): ** Told not to do until target is selected


 * GC% Content for gene (codon optimized): ** Told not to do until target is selected


 * PDB # or closest PDB entry if using homology model: ** 10.2210/pdb1ile/pdb (1ILE) []

(target from different source organism that is similar in structure according to PDB: [] ; source organism is Thermus thermophilus)


 * For Homology Model option: ** []


 * Show pairwise alignment of BLASTP search in NCBI against the PDB: ** []

Query 1 MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA 60 MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA Sbjct 1 MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA 60

Query 61 RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR 120 RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR Sbjct 61 RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR 120

Query 121 ESVFTYEKEWEAFTERIAYWVDLEDAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY 180 ESVFTYEKEWEAFTERIAYWVDLE+AYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY Sbjct 121 ESVFTYEKEWEAFTERIAYWVDLENAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY 180

Query 181 CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA 240 CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA Sbjct 181 CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA 240

Query 241 AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTQVLKTFPGKALEGLPYTPPYPQALEKG 300 AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGT VLKTFPGKALEGLPYTPPYPQALEKG Sbjct 241 AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTPVLKTFPGKALEGLPYTPPYPQALEKG 300

Query 301 YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR 360 YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR Sbjct 301 YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR 360

Query 361 EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRNNQ 420 EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIR NQ Sbjct 361 EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRKNQ 420

Query 421 EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA 480 EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA Sbjct 421 EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA 480

Query 481 TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR 540 TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR Sbjct 481 TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR 540

Query 541 ESFPADFIAEGIDQTRGWFNSLHQLGVMLFGSIAFKNVICHGLILDEKGQKMSKSKGNVV 600 ESFPADFIAEGIDQTRGWFN LHQLGVMLFGSIAFKNVICHGLIL E GQKMSKSKGNVV Sbjct 541 ESFPADFIAEGIDQTRGWFNFLHQLGVMLFGSIAFKNVICHGLILYEMGQKMSKSKGNVV 600

Query 601 DPWDIIRKFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD 660 DPWDIIR+FGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD Sbjct 601 DPWDIIREFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD 660

Query 661 RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV 720 RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV Sbjct 661 RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV 720

Query 721 RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRLEAKES 780 RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVR EAKES Sbjct 721 RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRPEAKES 780

Query 781 VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV 821 VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV Sbjct 781 VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV 821


 * Query Coverage: ** 100%


 * Max % Identities: ** (813/821) 99%


 * % Positives: ** (815/821) 99%

MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQARSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACRESVFTYEKEWEAFTERIAYWV DLENAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPYCPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAAAVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTPVLKTFPGK ALEGLPYTPPYPQALEKGYFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFREANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRKNQEIH WVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARATKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFRESFPADFIAEGIDQTRGWFNFLHQ LGVMLFGSIAFKNVICHGLILYEMGQKMSKSKGNVVDPWDIIREFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLDRPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSAR ALRDFVVEDLSQWYVRRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRPEAKESVHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGVKTRTPLPLLLVTAPTALEREGLKRF AHEIAEELNVKEVRVLEPGEEILSYRVLPNLKLLGRKYGKLVPKIREALQRERERAAALALKGEAIPLEVEGEALTLLPEEVLLEAEAPKGYQALEKDGYVAALKVEVTEALRMEGLARDLIRLLQQARKDMGLKVSDRIR VGYEAEGPYLEALKRHGPWIAEEVLATAFGEGLFGGFEARVEDEEGKAVFHLARAE
 * Chain used for homology: **


 * Primer design results for 'tail' primers (this is just 2 sequences): ** Told not to do until target is selected