TargetSp17+- NH3-dependent+NAD synthetase+(Clostridium+botulinum)


 * *Target (protein/gene name): ** NH3-dependent NAD+ synthetase (nadE)
 * *NCBI Gene # or RefSeq#: **5400613
 * *Protein ID (NP or XP #) or Wolbachia#: **YP_001386356.1
 * *Organism (including strain): **//Clostridium botulinum A str. Hall//
 * Etiologic Risk Group (see link below): ** RG2
 * */ Disease Information (sort of like the Intro to your Mini Research Write up): **Botulism is a paralytic disease that is caused by the nerve toxins of Clostridium botulinum and can be contracted through consuming the toxin in poorly prepared foods (especially canned foods) or infection of a wound. Early symptoms, such as double vision, drooping eyelids, and muscle weakness, can lead to paralysis of limbs or respiratory muscles if left untreated. Due to botulism's similarity to other diseases, like Guillain-Barré syndrome and myasthenia gravis, brain scans or spinal fluid examinations must be done to ensure that a patient has it. Victims of the disease can be placed under medical care or be administered an antitoxin, both of which can alleviate the symptoms and have reduced the fatality rate of the disease from 50% to 3-5% over the past decades.

Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): dimer [] []
 * Link to TDR Targets page (if present): ** N/A
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **[]
 * Essentiality of this protein: ** NH3-dependent NAD+ synthetase is an enzyme that produces the cofactor NAD+, which is used in a variety of cellular processes. Inhibition of the protein causes the failure of cell metabolism.
 * Complex of proteins?: ** N/A
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): ** Virtual screening was performed on nadE crystal structures from //B. anthracis // and //B. subtilis //and revealed several commerical compounds (5617, 5824, and 5833) that inhibit the enzyme's activity.
 * *EC#: 6.3.1.5 **
 * Link to BRENDA EC# page: **
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic


 * Figure 1.** BRENDA mechanism schematic of the NAD+ synthesis performed by NH3-dependent NAD+ synthetase

Sigma Assay sheet - DR. B) Text S1 from the paper [|http://mbio.asm.org/content/5/1/e00747-13.full#F5] -ATP: FLAAS SIGMA,[|34369-07-8], 1 mg, $36.70 -HEPES pH 7.4: H0887 SIGMA, [|7365-45-9], 20 ml, $16.50 -Ammonium chloride: A9434 SIGMA, [|12125-02-9], 500 g, $25.60 -Semicarbazide: 363634 ALDRICH, [|57-56-]7, 25 g, $51.90 -Ethanol: 652261 SIGMA-ALDRICH, [|64-17-5], pricing unavailable -Magnesium chloride: 1374248 USP, [|7791-18-6], 1 g, $331.00 -Alcohol dehydrogenase: 55689 SIGMA, [|9031-72-5], 100 mg, $99.10
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): ** continuous and discontinuous coupled enzyme assays, direct high-performance LC (HPLC) analysis - use the spectrohpotometric assay only - Dr. B (
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **
 * -- links to assay reagents (substrates) pages. **
 * --- List cost and quantity of substrate reagents, supplier, and catalog # **

-- PDB #: 1XNG MQKDYQKLIVYLCDFLEKEVQKRGFKKVVYGLSGGLDSAVVGVLCQKVFKENAHALLMPSSVSMPENKTDALNLCEKFSI PYTEYSIAPYDAIFSSHFKDASLTRKGNFCARLRMAFLYDYSLKSDSLVIGTSNKSERMLGYGTLFGDLACAINPIGELF KTEVYELARRLNIPKKILNKPPSADLFVGQSDEKDLGYPYSVIDPLLKDIEALFQTKPIDTETLAQLGYDEILVKNITSR IQKNAFKLELPAIAKRFNPELEHHHHHH
 * Structure (PDB or Homology model) **
 * Current Inhibitors: ** Gentamicin
 * Expression Information (has it been expressed in bacterial cells): **The protein has been expressed in M. tuberculosis H37Rv (Rv2421c)
 * Purification Method : **<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">metallo-affinity, FPLC size-exclusion chromatography
 * Image of protein (PyMol with features delineated and shown separately): **
 * Figure 2.** PyMol representation of NH3-dependent NAD+ synthetase (PDB#: 1XNG)
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **
 * Figure 2a. ** Blastp comparison of given amino acid sequence above to amino acid sequence from ncbi for NAD synthetase in //C. botulinum.//
 * *length of your protein in Amino Acids: ** 268
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 30,334.04 Da
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 19,620 M^-1cm^-1
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.


 * Figure 3.** TMpred graph for NH3-dependent NAD+ synthetase

<span style="background-color: #ffffff; font-family: monospace,serif;"><span class="ff_line">ATGAATAATTCACAATATTACATAAGAGTATCTACTGCTTGTCCTGTAACTAATGTGGCAGATATAGATT TTAATATAGACAACATAAAAAAGTGTATAGATATATGTATAGAAAAAAAATCAAAATTAATAGTTTTTCC GGAACTTTGTATAACTTCTTATACTTGTGGAGAACTTTTTTCACAAAGCCTATTAATATCCAAAGCTCTA GATGGAATACATAATATATGTAAATACTCTATAGATAAAGATGTTTTAATAGCTATAGGATCACCTTTAT TATATAAAAATAGTCTTTATAATTGTGCTGTTGTAATATTTGGAGGAAAAATACTAGGTATTGTCCCTAA AAGTTACTTACCTAATTATTCAGAGTTCTATGAGAAAAGATGGTTTACTGAAGGATATAAAATAAAAAGT GAAAGAATTAATTTACCATTCCAAGAGAACATTCCTTTTGGAGTTAACCTTATTTTTTCTGATAAAATTT TCAAATTTGCTTTCGAAGTATGTGAAGATCTTTGGGTTACAATTCCACCTAGTAGTTATCTTGCATTAAT GGGAGCTAATATTATTGGAAATTTATCTGCCTCTAATGAAATAGTTAGTAAATCCGATTATAGACGAAAT TTAGTTGCATCTCAAAGTGGTAGATGCTTAGCATCTTATGTATATTCCTCATCAGGGGTATACGAGTCTT CTACGGATTTAGTATTTAGCGGACACTTATTAATTGGAGAAAATGGCTCTATATTAAAAGAAAACAAAAG ATTCCAAAGAGAAAATGAAGTTATTACAAGTATTATAGATATAGATAAAATTAATAGCGAAAGACTTAAA AATGTGAGCTTTACAGATAACTCAATGAATATGAACTTGGAATTAGAAGAAATAACATTTCAATTTGCAA TTAACGATGTTGGTGACTTTGATAGACCTATAGACAGATACCCTTTTGTTCCTTCTAATGAAGAAAAAAG AGCAGTAAGATGCAAAGAAATATTTAATATACAAACCTCAGCTCTAGCTAAAAGACTTGATCATACTAAT ATGAAAAAAGCAGTTATAGGTATCTCCGGTGGATTAGATTCTACCTTAGCTTTATTAGTTATAGCTAAAA CCTTTGATAAATTAAACATTCCTAGAGAAAACATAATAACTATAACAATGCCTGGTTTTGGAACTACAGA TAGAACTTATAATAATGCTGTAAGTCTATGTAAACACTTAAATACAACCTTAAAAGAAATAAATATAGTA GATGCAGCATTACAACATTTTAAAGATATAGGCCATGACAAGGATATACATGATGTTACCTATGAAAATG TTCAAGCAAGAGAGAGAACTCAAATACTTATGGATATAGCCAATAAAGAAGGTGGCTTAGTTATAGGAAC CGGTGATTTATCTGAATTAGCTTTAGGCTGGTGTACATATAATGGAGATCATATGTCCATGTACAGCGTA AATTGCTCTATTCCTAAGACCTTAGTAAGATATCTGGTGAGATATGTAGCTGAAAATGAAGTAGCAAAAG AAATATCAGAAATACTTATAGATATTTTAGATACACCTGTAAGTCCTGAACTGTTACCAAAGGATAAAGA GGGCAACATTGTTCAAAAAACTGAAGATATAGTAGGACCTTATGAGTTGCATGATTTCTTTCTTTATTAT TTTATTCGTCAAGGAGCTACTCCAGATAAAATAAAACAATTAGCTAAAATAGCTTTTAAAGATTCCTATG ATAAAGAAACTATTGACAAATGGTTCTCTTATTTTATAAGAAGATTCTTTACCCAACAATTTAAACGTTC CGCAGTACCAGACGGTCCTAAAGTTGGAACTATAAGTTTATCCCCTAGAGGAGATTGGAGAATGCCATCA GACGCAAGTTTTAACCTTTGGAAATAA
 * *CDS Gene Sequence (paste as text only): **
 * *GC% Content for gene: ** 29.99%
 * *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)

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 * Primer design results for 'tail' primers (this is just 2 sequences): **