Plasmodium+falciparum+Adenylosuccinate+Synthetase


 * ** Target (protein/gene name): **// Plasmodium falciparum // Adenylosuccinate Synthetase (PfAS)
 * ** NCBI Gene # or RefSeq#: ** NCBI Gene # 814251
 * ** Protein ID (NP or XP #) or Wolbachia#: ** XP_001350257.1
 * ** Organism (including strain): ** //Plasmodium falciparum//
 * ** Etiologic Risk Group (see link below): ** Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
 * ** Disease Information (sort of like the Intro to your Mini __Research Write__ up):** Plasmodium falciparum is a protozoan parasite accounting for the majority of the approximately one million worldwide deaths from malaria annually [1]. Because of P. falciparum’s increasing resistance to current drugs such as artemisinins [2], discovering novel drugs for this pathogen is crucial for maintaining effective malaria treatment and reducing mortality.
 * ** Link to TDR Targets page (if present): **http://www.tdrtargets.org/targets/view?gene_id=2266
 * ** Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **
 * **Essentiality of this protein:** P. falciparum adenylosuccinate (PfAS) catalyzes the synthesis of adenylosuccinate from inosine monophosphate (IMP) and aspartate as an intermediate step of the purine salvage pathway. Since P. falciparum cannot synthesize purines through any other means, PfAS is an essential protein for P. falciparum, and disrupting its function would hinder P. falciparum from synthesizing nucleic acids and replicating DNA, impeding its reproduction in the host [3].
 * ** Is it a monomer or multimer as biological unit? ** (make prediction at @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer
 * ** Complex of proteins?: ** No
 * ** Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): " ** In ancillary studies, it was shown for the first time in vivo that the parenteral administration of L-alanosine reduces the specific activity of intratumoral **adenylosuccinate ** **synthetase ** by 70% and depresses the synthesis of DNA to an equivalent or greater extent..." [4]


 * ** EC#: ** 6.3.4.4
 * ** [|Link to BRENDA EC# page] **
 * ** BRENDA enzyme mechanism schematic:[[image:PfAS reaction diagram.PNG width="970" height="275"]] **
 * ** Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **BIOMOL® Green free phosphate spectrophotometric assay
 * ** link to Sigma (or other company ) page for assay (see Sigma links below): ** [|BIOMOL® Green]
 * ** citation to paper that contains assay information: ** [5]
 * ** links to assay reagents (substrates) pages. ** [|GTP], [|IMP], [|L-aspartate]
 * ** cost and quantity of substrate reagents, supplier, and catalog #: ** see links above


 * Structure (PDB or Homology model) **
 * PDB ID: 1P9B [3]

> ATGAACATCTTCGACCATCAAATTAAGAATGTTGACAAAGGTAACGTGGTAGCGATCCTG GGTGCGCAGTGGGGTGACGAGGGTAAAGGCAAAATCATTGACATGCTGTCTGAATACTCT GACATCACTTGCCGTTTCAACGGTGGCGCGAATGCGGGTCACACCATCTCTGTTAACGAT AAGAAATACGCGCTGCACCTGCTGCCTTGCGGTGTTCTGTACGACAACAACATCTCCGTT CTCGGCAATGGTATGGTTATTCACGTTAAGTCTCTCATGGAGGAAATCGAGTCTGTTGGC GGTAAACTGCTCGACCGCCTGTACCTGTCTAACAAAGCGCACATCCTGTTCGACATCCAC CAGATCATCGATTCTATCCAGGAAACCAAAAAGCTGAAGGAAGGTAAACAAATCGGTACG ACCAAGCGTGGTATCGGCCCGTGCTACTCTACCAAAGCGTCTCGTATTGGTATCCGCCTG GGCACCCTCAAGAACTTTGAAAACTTCAAAAACATGTACTCTAAGCTGATTGACCACCTG ATGGACCTGTACAATATCACCGAGTATGACAAGGAAAAGGAGCTCAACCTCTTCTACAAC TATCACCTGAAGCTGCGTGACCGTATCGTTGACGTTATCTCTTTTATGAACACCAACCTG GAGAACAACAAGAAGGTACTGATCGAAGGTGCGAACGCGGCCATGCTCGATATTGACTTC GGTACCTACCCGTACGTGACGTCTTCTTGCACTACCGTTGGTGGTGTTTTCTCTGGTCTC GGTATCCATCACAAGAAACTGAACCTCGTTGTAGGTGTAGTCAAATCTTACCTGACCCGT GTTGGTTGCGGCCCATTCCTGACCGAACTGAATAATGACGTTGGTCAGTATCTCCGCGAG AAAGGTCACGAATACGGTACTACTACGAAACGTCCGCGTCGTTGTGGTTGGCTGGATATT CCGATGCTGCTGTACGTTAAATGCATCAACTCTATCGACATGATCAACCTGACGAAACTG GACGTTCTGTCCGGTCTCGAAGAGATTCTGCTCTGTGTTAACTTTAAGAACAAAAAAACC GGTGAACTCCTCGAAAAAGGCTGTTACCCAGTTGAGGAAGAAATCTCTGAAGAATACGAA CCTGTTTACGAGAAATTCAGCGGTTGGAAGGAGGACATCTCTACCTGCAATGAGTTCGAC GAGCTCCCTGAGAACGCGAAGAAGTACATTCTGGCAATCGAAAAGTACCTCAAAACCCCG ATCGTTTGGATCGGTGTAGGTCCGAACCGTAAGAATATGATCGTAAAAAAGAATTTCAAT CTGAACTAA >
 * ** Current Inhibitors: **
 * ** Expression Information (has it been expressed in bacterial cells): ** " Recombinant PfAdSS [PfAS] expressed in E. coli BL21 (DE3) was purified as reported" [3].
 * ** Purification Method : ** Nickel-NTA affinity chromatography
 * ** Image of protein (PyMol with features delineated and shown separately):[[image:PfAS substrates.png width="1014" height="610" caption="PfAS: C green, O red, N blueGTP: C pink, O red, N blue, P orange; IMP: C cyan, O red, N blue, P orange; Hadacidin: C yellow, O red, N blue; Mg2+: green sphere "]] **
 * ** Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **MNIFDHQIKNVDKGNVVAILGAQWGDEGKGKIIDMLSEYSDITCRFNGGANAGHTISVNDKKYALHLLPCGVLYDNNISVLGNGMVIHVKSLMEEIESVGGKLLDRLYLSNKAHILFDIHQIIDSIQETKKLKEGKQIGTTKRGIGPCYSTKASRIGIRLGTLKNFENFKNMYSKLIDHLMDLYNITEYDKEKELNLFYNYHLKLRDRIVDVISFMNTNLENNKKVLIEGANAAMLDIDFGTYPYVTSSCTTVGGVFSGLGIHHKKLNLVVGVVKSYLTRVGCGPFLTELNNDVGQYLREKGHEYGTTTKRPRRCGWLDIPMLLYVKCINSIDMINLTKLDVLSGLEEILLCVNFKNKKTGELLEKGCYPVEEEISEEYEPVYEKFSGWKEDISTCNEFDELPENAKKYILAIEKYLKTPIVWIGVGPNRKNMIVKKNFNLN
 * ** length of your protein in Amino Acids: ** 442 aa
 * ** Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 50.0648 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength: 53915, assuming all pairs of Cys residues form cystines; 53290, assuming all Cys residues are reduced
 * ** 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): ** ATGAACATATTTGATCATCAAATAAAAAATGTGGATAAAGGGAATGTAGTTGCAATATTAGGTGCACAATGGGGTGATGAAGGGAAAGGAAAAATAATTGATATGTTATCAGAATATTCTGATATTACTTGTAGATTTAATGGAGGTGCTAATGCAGGACATACGATATCAGTAAATGATAAGAAATATGCTTTACATTTATTACCATGTGGTGTATTATATGATAATAATATAAGTGTATTAGGAAACGGAATGGTAATACATGTAAAATCATTAATGGAAGAAATTGAATCAGTTGGGGGAAAGTTGTTAGATAGATTATATTTATCAAATAAAGCACATATATTATTTGATATTCATCAAATTATTGATTCAATCCAAGAAACGAAAAAATTAAAAGAAGGAAAACAAATAGGTACAACAAAAAGAGGTATTGGACCATGTTATTCTACTAAAGCTTCCAGAATAGGTATAAGATTAGGAACTTTAAAAAATTTTGAAAATTTTAAAAATATGTATAGTAAATTAATAGACCACTTAATGGATTTATATAATATAACAGAATATGACAAAGAAAAAGAACTCAACTTATTTTATAATTATCACTTAAAGTTAAGAGATAGAATAGTTGATGTTATTTCCTTTATGAATACAAATTTAGAAAACAACAAAAAAGTATTAATTGAAGGTGCTAATGCAGCTATGTTAGATATTGATTTTGGAACATATCCATATGTAACTAGTAGCTGTACAACAGTTGGTGGGGTTTTCTCAGGACTTGGAATTCATCATAAAAAACTGAATTTAGTTGTAGGTGTAGTTAAAAGTTACTTAACCAGAGTTGGATGTGGCCCTTTCTTAACTGAATTAAATAATGACGTTGGTCAATATTTAAGAGAAAAAGGTCATGAATATGGAACGACTACCAAGAGACCAAGAAGGTGTGGATGGCTCGACATACCAATGTTATTATATGTTAAGTGCATTAATAGTATTGATATGATAAACTTAACAAAATTGGATGTTTTATCTGGATTAGAGGAAATATTATTGTGTGTCAATTTTAAAAATAAAAAAACAGGTATCAATTAAAGCATATGTATATTTTATATGTGTAATATGTAAATATTATATCTTAATGTTTTCATATATATATATATATATATATATATACATTTATTTATTTATTTTTTTTTTTTTTTAGGAGAACTGCTTGAAAAGGGTTGCTACCCTGTTGAAGAAGAAATATCAGAAGAATATGAACCTGTTTATGAAAAATTCAGTGGATGGAAAGAAGACATCTCAACTTGTAATGAATTTGATGAATTACCAGAAAATGCAAAAAAATATATTTTAGCTATAGAGAAATATTTAAAAACTCCAATAGTTTGGATTGGTGTAGGTCCTAATAGAAAAAATATGATAGTTAAAAAGAATTTTAACCTAAACTAA
 * ** GC% Content for gene: ** 26.4%
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
 * ** GC% Content for gene (codon optimized): ** 47.1%

-- 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): ** see link below
 * ( [|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): **


 * References:**
 * 1) Murray, C. J. et al., Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet 2012, 379 (9814), 413-31.
 * 2) Takala-Harrison, S. et al., Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis 2015, 211 (5), 670-9.
 * 3) Eaazhisai, K. et al., Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum. J Mol Biol 2004, 335 (5), 1251-64.
 * 4) Tyagi, A. K.; Cooney, D. A., Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase. Cancer Res 1980, 40 (12), 4390-7.
 * 5) Myers, C. L. et al., Characterization of wall teichoic acid degradation by the bacteriophage φ29 appendage protein GP12 using synthetic substrate analogs. Journal of Biological Chemistry: 2015; p 5.