Target+-+Phosphoserine+phosphatase+(Vibrio+cholerae)


 * Target Team:** John G., Keenan W., Samantha D., Avery W.


 * Target (protein/gene name):** Phosphoserine Phosphatase


 * NCBI Gene # or RefSeq#:** 243277


 * Protein ID (Uniprot):** VC_2345


 * Organism (including strain):** Vibrio cholerae


 * PDB ID**: 3N28

http://www.rcsb.org/pdb/explore/explore.do?structureId=3n28
 * PDB Website:**


 * Etiologic Risk Group (see link below):** Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia

Cholera is an infection of the small intestine caused by the gram-negative bacterium Vibrio cholerae and characterized by clear diarrhea and vomiting so severe that is causes extreme dehydration and death in many patients. Cholera is sometimes termed the "blue death" because pa tients suffering from severe cases develop a blue tint to their skin as a result of such severe dehydration. Symptoms can begin anywhere from hours after ingestion of the bacteria to nearly five days after exposure.
 * Disease Information (sort of like the Intro to your Mini __Research Write__ up):**[[image:arw2645_vc_isolated.jpg width="176" height="143" align="right" caption="The bacterium Vibrio cholerae."]]

The Vibrio cholerae bacteria was first isolated in 1854 by the Filippo Pacini, an Italian anatomist. As of 2010, it affects 3-5 million people every year andclaims between 58,000 a nd 130,000 deaths per year, mostly in the developing world. The actualnumber of cases and/or deaths may be much higher than that reported, as many cases may go unreported out of fear that reports of cholera may negatively impact tourism in the affected countries.

Typically bacteria cannot survive the acidic conditions of the human stomach, but Vibrio cholerae can by shutting down protein production while inside the stomach to conserve their energy and nutrients. After they make it through the stomach, in response to the environmental change, the surviving bacteria begin to produce flagella to swim through the mucus lining the small intestine to reach the intestinal walls. Once they reach the walls, again as a response to a change in environment, they begin producing the toxic protein that causes diarrhea in patients and the next generation of bacteria are voided from the body to infect future victims (if waste is not properly sanitized).

The toxic protein Vibrio cholerae produces is called cholera toxin (CT or CTX) and is made up of six protein subunits (one A subunit and five B subunits) connected by a disulfide bond. The B subunits form a five-part ring around the A subunit and allows the toxin to bind to the epithelial walls of the small intestine. The toxin in taken into the epithelial cells via receptor-mediated endocytosis, and upon entry into the cell the disulfide bond connecting the A subunit to the B subunits is reduced. Part of the A subunit is released and becomes free to interact with the human protein Arf6 (the human equivalent of the protein that the subunit usually interacts with in the Vibrio cholerae cell). Binding to Arf6 exposes the active site of the A subunit and allows the toxin to permanently ribosylate the Gs alpha subunit of the human G protein, which causes constitutive cAMP production in the human cell. This causes to cell to secrete H 2 O, Na +, K + , Cl − , and HCO 3− and become severely and rapidly dehydrated. Dehydration becomes to severe so quickly, that when patients seek treatment they need something like ten percent of their body weight replaced with fluids in the first few hours.

The disease is usually spread by ingestion of contaminated food or water, typically being transmitted through seafood in the developed world and water in the undeveloped world. Cholera can only very rarely be spread from person to person - in underdeveloped parts of the world it is much more often spread as a result improper treatment of the diarrhea from infected individuals so the waste contaminates water supplies.The spread of the disease can pretty generally be prevented by proper sanitation of waste and any other supplies that have come into contact with the patient (bedding, clothes, etc) and proper purification of water by boiling, chlorination, of even simply cloth filtration in areas where the other techniques are not possible.

The best way to treat cholera as of yet is through oral rehydration therapy which replenishes water and electrolytes in the body - antibiotics are used only in patients with particularly severe dehydration, as all antibiotics can do is shorten the course of the disease. Typically doxycycline is used, although some strains of Vibrio cholera have become resistant. There are some oral vaccines available for cholera, although many of them have limited availability. The best is Dukoral, which has minimal side effects and is 52% effective in the first year after ingestion and 62% effective in the second year. It is available in many countries, but generally not recommended by the CDC for people from the United States traveling to affected countries. The World Health Organization recommends vaccination of only high-risk groups (small children, people with HIV, etc) out of concern that mass vaccination could lead to Vibrio cholerae developing immunity to all known treatments.

N/A
 * Link to TDR Targets page (if present):**


 * EC#: **3.1.3.3.


 * Link to BRENDA EC# page:** []
 * --** Show screenshot of BRENDA enzyme mechanism schematic
 * Image of protein:**




 * Gene Sequence (derived from oligo): **

ATGTCTCTGGACGCGCTGACGACCCTCCCAATCAAAAAACACACCGCACTGCTCAACCGC TTCCCGGAGACCCGTTTCGTTACCCAGCTCGCGAAAAAACGTGCGTCTTGGATCGTTTTC GGTCACTATCTCACCCCAGCGCAATTCGAAGACATGGACTTCTTCACCAACCGTTTTAAC GCGATCCTGGACATGTGGAAAGTTGGTCGTTACGAAGTTGCGCTCATGGACGGTGAACTG ACCTCTGAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTATGCGCGTATTCAGGAC GTTCCGGACCTGACTAAGCCTGGTCTGATCGTTCTGGATATGGATTCTACCGCGATTCAA ATCGAATGCATCGACGAAATCGCAAAACTGGCGGGTGTAGGTGAGGAGGTTGCGGAAGTT ACCGAGCGTGCAATGCAGGGCGAACTGGACTTTGAACAGTCTCTGCGTCTCCGTGTTTCT AAGCTCAAGGATGCGCCTGAACAGATCCTGTCTCAAGTTCGTGAAACCCTGCCACTGATG CCGGAACTGCCGGAGCTGGTGGCGACCCTGCACGCGTTCGGTTGGAAGGTTGCCATCGCG TCTGGTGGTTTTACCTACTTCTCTGACTACCTGAAGGAACAACTCAGCCTGGACTACGCT CAGTCTAACACCCTCGAAATTGTTTCTGGTAAACTGACTGGTCAGGTTCTCGGTGAGGTT GTTAGCGCGCAAACCAAAGCAGACATCCTGCTGACCCTGGCGCAGCAATACGACGTTGAA ATCCACAATACCGTTGCGGTTGGCGATGGTGCCAACGACCTGGTTATGATGGCCGCAGCC GGTCTGGGTGTTGCGTATCACGCTAAACCTAAGGTTGAGGCGAAGGCCCAAACGGCGGTT CGTTTCGCTGGCCTGGGCGGTGTTGTTTGCATTCTGTCCGCTGCGCTCGTCGCGCAACAG AAACTGAGCTGGAAATCTAAACCGTAA

1 ATGTCTCTCGATGCGCTGACCACCCTCCCGATCAAAAAACACACCGCACTGCTGAACCG 59 2 CACGCTTCTTCGCGAGCTGGGTAACGAAACGAGTCTCTGGGAAGCGGTTCAGCAGTGCGG 60 3 GCTCGCGAAGAAGCGTGCGTCTTGGATTGTTTTCGGTCACTATCTCACTCCGGCGCAGTT 60 4 GAGGATCGCGTTAAAACGATTGGTGAAAAAGTCCATATCTTCGAACTGCGCCGGAGTGAG 60 5 AATCGTTTTAACGCGATCCTCGATATGTGGAAAGTTGGCCGTTACGAGGTGGCCCTGATG 60 6 GCGCTTTCAGGATGGTTTCGTGTTCAGACGTCAGTTCACCGTCCATCAGGGCCACCTCGT 60 7 GAAACCATCCTGAAAGCGCTGGAACTGGATTATGCGCGTATCCAGGACGTTCCGGACCTG 60 8 TCGCGGTAGAGTCCATGTCCAGCACGATCAGGCCCGGTTTAGTCAGGTCCGGAACGTCCT 60 9 ACATGGACTCTACCGCGATTCAGATCGAATGCATCGACGAAATCGCGAAACTGGCGGGTG 60 10 ACCTTGCATGGCACGCTCGGTGACTTCCGCAACCTCCTCGCCAACACCCGCCAGTTTCGC 60 11 GCGTGCCATGCAAGGTGAGCTGGATTTTGAACAGTCTCTCCGCCTCCGCGTGTCTAAGCT 60 12 GTTTCACGAACCTGGCTCAGGATCTGTTCTGGCGCGTCTTTGAGCTTAGACACGCGGAGG 60 13 TGAGCCAGGTTCGTGAAACCCTGCCGCTCATGCCGGAACTGCCGGAGCTCGTTGCTACTC 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">14 AGCCACCAGAGGCGATGGCAACCTTCCAACCGAACGCGTGCAGAGTAGCAACGAGCTCCG 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">15 CATCGCCTCTGGTGGCTTCACCTACTTCTCTGACTACCTGAAGGAACAGCTCTCTCTGGA 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">16 TTTACCAGAAACGATCTCCAGGGTGTTAGACTGCGCATAGTCCAGAGAGAGCTGTTCCTT 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">17 CCTGGAGATCGTTTCTGGTAAACTGACTGGTCAGGTGCTGGGTGAAGTTGTTTCCGCGCA 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">18 TCGTACTGCTGCGCCAGGGTCAGGAGGATGTCCGCTTTGGTCTGCGCGGAAACAACTTCA 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">19 TGGCGCAGCAGTACGATGTTGAAATCCACAACACCGTTGCGGTAGGCGACGGTGCGAACG 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">20 CGTGGTACGCAACGCCGAGGCCGGCTGCTGCCATCATAACCAGGTCGTTCGCACCGTCGC 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">21 GGCGTTGCGTACCACGCCAAGCCGAAAGTAGAAGCGAAGGCGCAAACGGCGGTACGCTTC 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">22 CGAGGGCCGCAGACAGGATGCAAACCACACCACCGAGACCAGCGAAGCGTACCGCCGTTT 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">23 CTGTCTGCGGCCCTCGTAGCGCAACAAAAACTCAGCTGGAAATCTAAAGAGGGTCATCAC 60 <span style="display: block; font-family: 'Courier New',Courier,monospace; text-align: left;">24 TTAATGATGATGGTGGTGATGACCCTCTTTAGATTTCC 38
 * Primers Ordered:**


 * Length of your protein in Amino Acids: ** 328 Amino Acids

First 5 residues (MSALD) from PDB replaced first 5 of Uniprot sequence. http://www.rcsb.org/pdb/explore/explore.do?structureId=3n28 http://www.uniprot.org/uniprot/Q9KPM2
 * Protein Sequence:**

<span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium;">MSLDALTTLPIKKHTALLNRFPETRFVTQLAKKRASWIVFGHYLTPAQFEDMDFFTNRFN AILDMWKVGRYEVALMDGELTSEHETILKALELDYARIQDVPDLTKPGLIVLDMDSTAIQ IECIDEIAKLAGVGEEVAEVTERAMQGELDFEQSLRLRVSKLKDAPEQILSQVRETLPLM PELPELVATLHAFGWKVAIASGGFTYFSDYLKEQLSLDYAQSNTLEIVSGKLTGQVLGEV VSAQTKADILLTLAQQYDVEIHNTVAVGDGANDLVMMAAAGLGVAYHAKPKVEAKAQTAV <span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium;">RFAGLGGVVCILSAALVAQQKLSWKSKP


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: **83546.8


 * Extinction Coefficient:** 11170 M -1 cm -1


 * GC-Content**: t0.374532


 * TMpred Graph Image:**

__ Forward Primer: __ 5’ __ TACTTCCAATCCATGTCTCTGGACGCG __ 3’ 27 bp GC Content 51.9% 0.0 mM Mg2+ Tm 62.3 oC 1.5 mM Mg2+ Tm 68.8 oC  **2.0 mM Mg2+ Tm 69.3 oC** 4.0 mM Mg2+ Tm 70.4 oC 6.0 mM Mg2+ Tm 70.9 oC
 * Primer Tail Design: **

__ Reverse Primer __ : 5’ __ TATCCACCTTTACTGTTACGGTTTAGATTTCCAGCT __ 3’ 36 bp GC Content 38.9% 0.0 mM Mg2+ Tm 61.8 oC 1.5 mM Mg2+ Tm 69.5 oC  **2.0 mM Mg2+ Tm 70.0 oC** 4.0 mM Mg2+ Tm 71.0 oC 6.0 mM Mg2+ Tm 71.5 oC

<span style="display: block; font-family: 'Times New Roman',Times,serif; font-size: 120%; text-align: center;">The primers listed below (in red) are what we originally ordered, but they're wrong. The forward primer needed eight base-pairs cleaved off the right end to bring it's melting temperature down to within a degree of the reverse primer. On the reverse primer, the last codon on the right side was never reverse complemented so it would not have been able to bind to our gene. 5’ TAC TTC CAA TCC ATG TCT CTG GAC GCG CTG ACG AC3’ 35 bp GC Content 54.3% 0.0 mM Mg2+ Tm 67.4 oC 1.5 mM Mg2+ Tm 74.0 oC  2.0 mM Mg2+ Tm 74.4 oC  4.0 mM Mg2+ Tm 75.2 oC  6.0 mM Mg2+ Tm 75.5 oC
 * Forward Primer: **

5’ TAT CCA CCT TTA CTG TTA CGG TTT AGA TTT CCA AGC 3’ 36 bp GC Content 38.9% 0.0 mM Mg2+ Tm 61.2 oC 1.5 mM Mg2+ Tm 68.9 oC  2.0 mM Mg2+ Tm 69.4 oC  4.0 mM Mg2+ Tm 70.4 oC  6.0 mM Mg2+ Tm 70.8 oC
 * Reverse Primer: **