Target-+dihydrofolate+reductase-thymidylate+synthase+(Leishmania+major)

dihydrofolate reductase-thymidylate synthase
 * Target (protein/gene name):**

Leishmania Major can cause a disease called Zoonotic cutaneous leishmaniasis which can occur among people who live in rural areas and is distinguished by skin lesions that can become inflamed. Necrosis sores may develop followed by hypersensitivity and involvement of immune complexes. This is part of the healing process and contributes to strong specific immunity. Though Zoonotic cutaneous leishmaniasis has been known to heal spontaneously, a new drug called miltefosine has been tested as efficient and safe in several clinical trials.
 * NCBI Gene # or RefSeq#:** TDR Targets ID: 25954
 * Protein ID (NP or XP #) or Wolbachia#:** ID: [|TriTrypDB / LmjF06.0860] [|GeneDB / LmjF06.0860]
 * Organism (including strain):** Leishmania major
 * Etiologic Risk Group (see link below):** Risk Group 2 (Parasitic Agents)
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**

African trypanosome)
 * Essentiality of this protein:** 6 days (this was for parasitic

Moreover, thymidine addition to the culture media did not help in obtaining null parasites, suggesting that the //dhfr-ts// gene may be essential for //T. cruzi// survival //in vitro//. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236718/).


 * Complex of proteins?** No
 * Druggable Target: 1**

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3
 * *EC#: ** 1.5.1.3
 * Link to BRENDA EC# page:**


 * --** Show screenshot of BRENDA enzyme mechanism schematic

Enzyme concentration was determined spectrophotometrically by following absorbance at 280nm. The extinction coefficient for //P. falciparum// TS-DHFR is 83740 M−1cm−1, and for //L. major// TS-DHFR is 69955 M−1 cm−1. DHFR steady-state activity was assessed by reacting enzyme with H2folate and NADPH, and following absorbance at 340 nm, which decreases **as** NADPH is depleted to form NADP+. An extinction coefficient of −12.8 mM−1 cm−1 was used for the DHFR reaction.
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**

http://www.brenda-enzymes.org/literature/lit.php4?e=1.5.1.3&r=685197


 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/dihydrofolatereductase.pdf


 * -- link (or citation) to paper that contains assay information**

http://www.jbc.org/content/272/21/13883.long


 * -- List cost and quantity of substrate reagents and supplier**

Tris-HCl-$49.00 (100g)---T5941 NADPH-$99.90 (25mg)N5130 Sodium Acetate--$44.10 (500g)S8750 Sigma Aldrich Supplier

-- PDB # or closest PDB entry if using homology model:
 * Structure Available (PDB or Homology model)**
 * Structure Available (PDB or Homology model)**

Chain A, Trypanosoma Cruzi Dihydrofolate Reductase-Thymidylate Synthase Complexed With Nadph, Dump And C-448 Antifolate

-- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB

Query Coverage: 98% Max % Identities: 348/523(67%) % Positives: 403/523(77%) Chain used for homology: Chain A

10-PROPARGYL-5,8-DIDEAZAFOLIC ACID METHOTREXATE NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE 2'-DEOXYURIDINE 5'-MONOPHOSPHATE However, due to its weaker contribution (relative to DHFR-TS; Table [|III]), PTR1 overexpression by gene amplification is apparently necessary to provide sufficient activity
 * Current Inhibitors:**
 * Expression Information (has it been expressed in bacterial cells):**

MSRAAARFKIPMPETKADFAFPSLRAFSIVVALDMQHGIGDGESIPWRVPEDMTFFKNQT TLLRNKKPPTEKKRNAVVMGRKTWESVPVKFRPLKGRLNIVLSSKATVEELLAPLPEGQR AAAAQDVVVVNGGLAEALRLLARPLYCSSIETAYCVGGAQVYADAMLSPCIEKLQEVYLT RIYATAPACTRFFPFPPENAATAWDLASSQGRRKSEAEGLEFEICKYVPRNHEERQYLEL IDRIMKTGIVKEDRTGVGTISLFGAQMRFSLRDNRLPLLTTKRVFWRGVCEELLWFLRGE TSAQLLADKDIHIWDGNGSREFLDSRGLTENKEMDLGPVYGFQWRHFGADYKGFEANYDG EGVDQIKLIVETIKTNPNDRRLLVTAWNPCALQKMALPPCHLLAQFYVNTDTSELSCMLY QRSCDMGLGVPFNIASYALLTILIAKATGLRPGELVHTLGDAHVYRNHVDALKAQLERVP HAFPTLIFKEERQYLEDYELTDMEVIDYVPHPAIKMEMAV
 * Purification Method:**
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**


 * length of your protein in Amino Acids:** 520aa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website** 58688.6

Ext. coefficient 69955 Abs 0.1% (=1 g/l) 1.192, assuming all pairs of Cys residues form cystines
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**


 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * CDS Gene Sequence (paste as text only):**

ATGGCTTCTACTGGTGAAACTATATGTAACAATGGCCAAAGCGCAGTTTCAGTAACAAGGAGGAGGAGTT ACCAAGTTGTAATAGCGGCGACAAGAGACATGGGTCTTGGTATGGACATGAAACTTCCTTGGGATCTACC TTCAGAGTATCAGTTTTTCCAAGATGTTACAACTAGGACATCCGATCCCACGAAGAGGAACGCGACTATA ATGGGAAGAAAATCATGGGAATCTACTCCTCTAGAGATTCGTCCTCTTCCTGGTCGGCTCAATATTGTCT TGACAAAGTCTAGCTGCCACAACATTGCCATTGATGAGAATGTACTGGTGTCTAGTAGCATGGAATCGGC TCTTGAACTTCTAGCCACTGAGCCCTATTCCTTGTCCATTGAGAAGGTCTTTGTCATAGGAGGCGGCGAG TTGTTAAGGTTTGCATCTTCTTTCCACAAGGGTTTTGTTCTTTTTCTAAAAACACATGTTTATGTCTTGA TATAAGCGTGTCATGCGATGTTTGAGACTTTAGACCTTGAGGTTTGATGTCTTGATTTGATTGATGGTAC AATGATTGAAGTTCTTTTAAGGCCTTAAGTTAGATGGTTGAAGTTTAGTACTGTGAATGAGAGAATGATT AATTGTTGTGGTTTCTCTACAGGAATTATATGAATGCATCCATCTGTGATGCTATCCACCTAACCGAAAT CGATATAAGCGTGCCATGTGACGCATTTGCACCGAGAGTGGATACTTCTCTGTACCGTCCGTGGTACTCA TCATTTCCAGTTGTGGAAAATGGAATTCGGTATAGTTTCAACACTTATGTCCGAAGAAAAGATGCCATTG TTGGCTCCGGTGAAAAGAAAAGTGTTGCTGAGTCAGATTTGAAGGAGTACTCGTTTTTGCCTAAGATGGT TTTCGAGAGGCATGAGGAGTTTGGTTACTTGAATCTTGTTCAAAACATTATATCTAGTGGAGACATGAAT GACAATAGTACTCTTTCTAAATTTGGCTGTCAGGTGAGAGCTACACTCAATCTTTGTTAGTATCTTTCTC CTTTAATTGAGCTTACATGTTCTTTGTTCTTGATTTGGTTCATTTACAGATGCGGTTCAATCTGCGCAAG ACTTTCCCGCTTCTCACGACCAAGGCATGTCTTCTTTTATTCACATCTCTTATCAAAGTAGTTCACCTTT ATTTGATCACAGAGATAAATTTTGTCGGTTTTTGTACCCGTTGCCTTTCAGAAAATATTCTGGCTTGGTG TTGTAGAGGAAATACTACAACTTATCAGTGGTTCAAACAATCCCAAGGTAAAACCAAGAGAGACCTGTGC TACTACACAGTAAATGACAATGATCATGCCCAAAATTCTTGGGTTAAAGATATGTTTTCTTTGTTTGATA TATTGATAAAAAACCGAGAATGTAACATACTAGATTTTAACCCGCGGTACACCGCGGAGACAATTAATTT TTTAATATATATAAAAGTTTGCAAATTGTATCTATATATAAAATATTTTTATTTTAAAGTTTACAATTGT TATTAAATAATGTCCTGTCAAACTCGTCTCGTAAAAACCTATTTATTTTATTAATGTTGATTTTATTTAT GATATGTTTATAAATCATTGTATAAATATTTTGTAATTTTGTAGTAATTAACTGCTATCAAATATTTCTG CAGTTTGATGCTTATAATCGAATTGTTATAGTCATGAAAAAAGCAATATCAAAAATGATAATTTTGTAGT GTTTAAATGATATTAAATATTTTGGAAGTTTTATTTTAGTAACTAATTGTGTAGTTAATGTGGAGAGATT TTGGGAAGATAGTTAAATGTCAAATATTTAATTCGAAGATTTACTTTCTAATTATCAAAAATAATTATTA AATGTCAGTGGCAGCTACTGTAAATAAGTCCCAACTTGATGATTTATTTCACAAAATGGCTGCAAAAATG TATATATAGATTATATCTCATATGTTCTTTCAGGAAAACGGTAGTCATATATGGGACACCGATGAGGCAA AAGAATATCTTGACAGGCAAGCACAAACTTTAGAATTTAAAATGCACTTTTACTTATACAAAACTCACAT CCATGCCTATGTTCCAGTTTCGGAGTGAATGCCACCGAAGAAGATGGAGACAACCCATTCTTGCATGGAC TTCACTGGAAACATTGTGATGCTAGGTTTGTGATGTCTCTTCTTTGGATGTTTGTCTCATATGCTGTGCC TCTTTTGACTGATTGGTTTTACATTTTACCAGTCAAGAATTCAGTCAGCTCTCTGATGTTATAAACAAAA TAAAGAACAATCCTCATGATCAAAGGATCATGCTTGCCGCTTGTAATCCATTAGATTTCAAGTTGTCGGT ATCTCCTTGTCATACGTTCACACAGGTTAGCACGTTTTTCATCTTGAAACTTGTTACATTTCCATTGTAA AATCATTTCAACGTTTCAAGATATACAAAACTGAAAATGATTTGGTTTTTGGTTGTTGTTGCCAGTTCTA TGTGGCAAATGGTGAAGTTTCTTGTCAAATATACCAAAGCTCAACCGAAGCGAGTATCGGGATTCCTTTC AGTATCGCTACATACTCTCTTTTGACATGCATCATCGCTCATGTTTGCGGTATGTACACATTTTATTAGC TCTTGAGTCTAAAGTAAAAGAGTTGAAACCTTTGTATGGTTTCTTGTCTTGACAGATCTTGGAGCTGGTG ATTTCATCCATGTGATTGGACAAGCCTATATTAACAAAGCTCACGTCAAGGCTATACAAAAACAGCTTCA AATCTCCCCTAAACCCTTCCCGGTAAGTACTAAATATATTTTGCTCTCTCTCAAATCAGGAAACAATATA ACTTGTGTGTCTCTAATGTATTAACGGTAAAAACAGATTTTGAAAATCAACCCTGAGAAAAAGAAGATGG ACAACTTTGAGGCCTCTGATTTGGAACTCATGAGGATATAA


 * GC% Content for gene: 36%**
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**
 * GC% Content for gene (codon optimized):**

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * ( link to DNA Works output text file - **that should be saved in your Google Docs folder after you did the primer design protocol)
 * Primer design results for 'tail' primers (this is just 2 sequences):**


 * Sources:**

NIAID Emerging and Re-emerging Infectious Diseases – Group I, II, and III [] NIAID Biodefense – Group III: Category A, B, and C Priority Pathogens [] TDR targets webpage [|www.tdrtargets.org] J Craig Venter Institute – New Infectious Diseases Targets [] Neglected and Tropical Diseases (PLoS One) - [] [] []


 * EUPATH ** : Eukaryotic Pathogens Database Resources: []

Etiologic Risk Group Categories (for pathogens):

__ http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 __

BRENDA webpage for enzymes – can search by substrate by doing advanced search []

SIGMA - chemical supplier- []

[] - Protein Similarity


 * CHECK CURRENT INHIBITORS **
 * Binding DB []