Target+Sp-15-+HPPK-DHPS,+2-amino-4-hydroxy-6-hydroxymethyldihydropteridine+pyrophosphokinase+(Francisella+Tularensis)

Target Sp-15- HPPK-DHPS <2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase>  ………………..Researcher: Kylie Johnson


 * Target (protein/gene name)**: HPPK-DHPS (2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase)


 * NCBI Gene # or RefSeq#:** folK FTT_0942c (gene name) 3191528 (gene ID)


 * Protein ID (NP or XP #) or Wolbachia#:** Mmdb_id: 121548


 * Organism (including strain):** Francisella tularensis (F. tularensis tularensis)


 * Etiologic Risk Group (see link below):** Category A NIAID Biodefense Research

Tularemia (AKA Pahvant Valley plague, rabbit fever, deer fly fever, and Ohara's fever) is caused by Francisella Tularensis, a gram negative bacteria. Disease found in Rabits, Hairs, Pikas, small rodents, and Humans. Spread by ticks, deer flies, some arthropods, and ingestion of contaminated water or food. The incubation period for the disease is 14 days, clinical symptoms in humans appear after 3 and include: fever, lethargy, loss of appetite, signs of sepsis, enlargement of the lymphnodes (similar to the bubonic plague) and possibly death. Overall fatality for untreated cases is 7%, however some strains are more virulent.
 * Disease Information (sort of like the Intro to your Mini Research Write up):**


 * Link to TDR Targets page (if present):** NA

[]
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)**

Statement of essentiality: “The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis.” From: []
 * Essentiality of this protein:**


 * Is it a monomer or multimer as biological unit? (make prediction at []):** Monomer


 * Complex of proteins?:** NA


 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** Examined in E. Coli, so druggable in E. Coli. From article found at link: []


 * EC#:** 2.5.1.15

Figure 1: Reaction schematic catalyzed by enzyme HPPK/DHPS
 * Link to BRENDA EC# page:** []


 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** coupled, enzymatic spectrophotometric assay


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

Final concentrations of reagents in the standard assay were as follows: 50 mM Tris–HCl (pH 8.5), 1.0 mg/ml bovine serum albumin, 5.0 mM MgCl2, 20 mM β-mercaptoethanol, 50 mM NADPH, 50 mM 6-hydroxymethyl-7,8-dihydropterin or the pyrophosphate, 50 mM p-ABA, and 1.0 μg/ml DHFR. ATP (final concentration 100 μM). Substrate: 4-aminobenzoate From: []
 * -- links to assay reagents (substrates) pages**

Couldn’t fine 4 aminobenzoate for sale, however Ethlyl4-aminobenzoate is marketable, esterhydrolisis could be done to remove the Ethyl. Cost: $29.20 Quantity: 5 grams Suplier: Sigma-Aldrich Catalog #: 112909
 * --- List cost and quantity of substrate reagents, supplier, and catalog #**


 * Structure (PDB or Homology model)**
 * -- PDB # or closest PDB entry if using homology model:** 4PVZ

Figure 2: PDB image of enzyme HPPK/DHPS, homology model: 4PVZ

HPPK/DHPS in plants feedback inhibited by dihydropteroate Methotrexate inhibits just DHPS Possibility of pterin based inhibitors Article looking into new class of inhibitors that targets the pABA binding sight to inhibit both HPPK/DHPS: []
 * Current Inhibitors:**


 * Expression Information (has it been expressed in bacterial cells):** Yes, in E.Coli


 * Purification Method:** metal affinity chromatography and ion exchange chromatography
 * Image of protein (PyMol with features delineated and shown separately):**

Figure 3: Pymol representation of HPPK/DHPS showing surface. color coded as carbon green Figure 4: Pymol Representation of HPPK/DHPS as cartoon. Carbon green. Oxygen red, and nitrogen blue.

MQYIIGIGTNIGFTIENIHLAITALESQQNIRIIRKASLYSSKAVLKEDAPKEWDIRFLNTAVKISSSLKPDELLVLLKD IELKIGRDLNAPAWSPRVIDLDILAAEDLILETDKLTIPHKELINRSFALAPLLELSKGWHHPKYVEWDLNIRLKELGEI VKLKQTLANTIRMGIVNLSNQSFSDGNFDDNQRKLNLDELIQSGAEIIDIGAESTKPDAKPISIEEEFNKLDEFLEYFKS QLANLIYKPLVSIDTRKLEVMQKILAKHHDIIWMINDVECNNIEQKAQLIAKYNKKYVIIHNLGITDRNQYLDKENAIDN VCDYIEQKKQILLKHGIAQQNIYFDIGFGFGKKSDTARYLLENIIEIKRRLELKALVGHSRKPSVLGLAKDSNLATLDRA TRELSRKLEKLDIDIIRVHKI
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**


 * length of your protein in Amino Acids:** 421

48243.9 Da
 * Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:**

44015
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**

Figure 5: TMPred graph of HPPK/DHPS enzyme.
 * TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html):**

GTGCAATATATTATAGGAATTGGAACAAATATTGGTTTTACTATCGAAAATATTCATCTTGCAATAACTG CACTAGAATCGCAACAAAATATAAGAATCATCAGAAAAGCAAGCTTATACAGTAGTAAAGCTGTCCTTAA AGAAGACGCTCCTAAAGAATGGGATATTAGATTTTTAAATACAGCTGTAAAAATTAGCTCTTCACTAAAG CCTGATGAACTTTTAGTACTCTTAAAAGACATAGAATTAAAAATAGGTAGAGACCTAAATGCTCCTGCAT GGTCACCTCGAGTAATTGATTTAGATATTCTTGCTGCTGAAGATCTAATTTTAGAAACAGACAAACTTAC TATTCCTCATAAAGAATTAATTAATCGTAGTTTTGCGTTGGCTCCTTTATTAGAATTATCAAAGGGTTGG CATCACCCTAAATATGTTGAATGGGATCTTAATATAAGATTAAAAGAATTAGGTGAGATAGTAAAACTTA AACAAACTCTTGCAAATACAATACGTATGGGTATAGTTAATCTGTCAAATCAATCTTTTTCAGATGGTAA TTTTGATGATAATCAACGTAAATTAAACCTTGATGAGCTAATACAAAGTGGTGCTGAAATTATTGATATC GGAGCTGAATCGACTAAGCCTGATGCCAAGCCTATATCGATTGAAGAAGAATTTAACAAATTAGATGAAT TCCTAGAATACTTTAAATCACAACTGGCAAATTTGATTTATAAACCATTAGTCAGTATTGACACACGCAA ACTAGAGGTAATGCAAAAAATTCTCGCTAAGCATCATGATATTATCTGGATGATAAATGATGTTGAATGT AATAATATTGAGCAAAAAGCACAGCTTATAGCTAAATATAATAAAAAGTATGTTATAATTCATAATTTGG GTATTACAGATAGAAATCAATATTTAGATAAAGAAAATGCTATAGATAATGTTTGTGATTATATTGAGCA AAAAAAGCAAATTCTTCTAAAACATGGTATAGCACAACAAAATATTTATTTTGATATTGGCTTTGGTTTT GGTAAAAAATCAGATACCGCTAGATACTTATTAGAGAATATCATCGAGATAAAAAGAAGATTAGAATTAA AAGCATTAGTTGGTCACTCACGTAAGCCATCAGTTTTAGGATTAGCAAAAGATAGTAATTTAGCAACTTT AGATCGAGCTACAAGAGAGCTTTCAAGAAAATTAGAGAAACTAGATATTGATATTATAAGAGTACACAAG ATTTAA
 * CDS Gene Sequence (paste as text only):**

29.69%
 * GC% Content for gene:**


 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized):**

(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- 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):**
 * Primer design results for 'tail' primers (this is just 2 sequences):**