TargetF15+-+D-alanine+Ligase+(Listeria+monocytogenes)

Listeriosis is a foodborne illness caused by infection with the bacterium Listeria monocytogenes. With a 21% case-fatality rate between 2009 and 2011 in the United States, listeriosis is the third highest cause of death of the major foodborne pathogens. Listeriosis is especially serious in unborn babies, newborn infants, pregnant women, elderly, and those with immunocompromised conditions and can result in fatal complications including miscarriages, septicemia, and meningitis. Listeria infection can result from eating unpasteurized milk products, raw produce, raw seafood, or improperly processed meat. Symptoms of listeriosis begin with fever, nausea, and diarrhea and progress to headaches, confusion, loss of balance, and convulsions. If symptoms persist and in high-risk patients, listeriosis is treated with ampicillin, often in combination with gentamicin, with the length of treatment varying by case [] [] [] []
 * *Target (protein/gene name): ** D-Alanine Ligase
 * *NCBI Gene # or RefSeq#: ** 985238
 * *Protein ID (NP or XP #) or Wolbachia#: ** NP_464381.1
 * *Organism (including strain): ** // Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) //
 * *Etiologic Risk Group: ** Risk group 2
 * * **** Disease Information: **
 * *Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): **
 * *Essentiality of this protein: **“D-Alanine ligase is involved in biosynthesis of peptidoglycan. In gram-positive bacteria, likely essential. Decrease of D-alanine ligase results in thinner cell walls and increased susceptibility to ampicillin when developing intracellularly.” http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900503/full
 * *Is it a monomer or multimer as biological unit? (make prediction at ** [] ): predicted dimer
 * *Complex of proteins **** ?: ** No
 * *Druggable Target **** (list number or cite evidence from a paper/database showing druggable in another organism): **


 * *EC#: ** 6.3.2.4
 * *Link to BRENDA EC# page: ** []
 * -- ** **Show screenshot of BRENDA enzyme mechanism schematic**

Spectrophotometric assay - Malachite Green/BIOMOL Green http://www.enzolifesciences.com/BML-KI102/1ml-phosphate-standard-800-microm-for-biomol-green/ http://www.enzolifesciences.com/BML-AK111/biomol-green/ BIOMOL Green, Enzo Life Sciences, Catalog No. BML-AK111-0250……..$123.00 (includes 250ml BIOMOL Green reagent and 800ul phosphate standard)
 * *Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * -- link to Sigma/company page for assay: **@http://www.enzolifesciences.com/BML-AK111/biomol-green/
 * -- -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 for Homology model: 2I80 -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB:
 * Structure **** (PDB or Homology model) **

Query Coverage: 99% coverage Max % Identities: 48% identity % Positives: 70% positive identity  Chain used for homology: A

 MKTKLILLYGGKSAEHEVSLQTAFSVINALDLEKFEAAPIYITNEGEWIQGPLLSGKLDF VEQLRFSATDTIKLATTESEKSEGEAISPAVLEADGQETVVFPLLHGPNGEDGTVQGLFE VLNIPYVGNGVLASSAAMDKIVMKKIFADAGIPQVPAVAVRLIDWKNYQEEMVAEMEEVL TYPVFVKPANLGSSVGISKATNKKELVDAMTEAFLYDRRVVVEQGVVAREIEMGVLGNDT PVCSVPGEILPEGAVATFYDYKAKYQDNNTALIIPTEVDPEILEQMKEYAIQAFLGLDAS GLVRADFFLTEDNQLFLNEVNTMPGFTPYSMYPLLWQETGLPYGALIERLVDLAKERHAA KNALKYKLED ATGAAAACTAAATTAATTTTATTATACGGTGGAAAATCTGCCGAACATGAAGTTTCATTACAAACAGCAT TTTCAGTTATCAATGCTTTGGATTTAGAAAAATTCGAAGCTGCGCCAATATATATTACGAATGAAGGTGA GTGGATTCAAGGACCACTTCTTTCTGGGAAGTTAGATTTTGTCGAACAATTACGTTTTTCGGCTACAGAT ACAATAAAACTTGCTACAACTGAATCAGAAAAATCAGAAGGGGAAGCAATTAGCCCGGCCGTTTTAGAAG CAGACGGACAAGAGACAGTCGTATTCCCACTTTTACATGGTCCAAACGGAGAAGATGGCACTGTTCAAGG ATTGTTCGAAGTATTAAACATTCCATATGTTGGCAACGGGGTCTTGGCATCTTCTGCTGCAATGGACAAA ATCGTGATGAAGAAAATTTTTGCAGATGCTGGAATCCCGCAAGTTCCAGCAGTTGCTGTTCGTTTAATTG ATTGGAAAAATTATCAAGAGGAAATGGTTGCTGAGATGGAAGAAGTCCTTACATATCCTGTCTTCGTAAA ACCAGCAAACCTTGGTTCGAGTGTTGGTATTAGTAAGGCAACAAACAAAAAAGAATTAGTGGACGCAATG ACAGAAGCGTTTTTATATGACCGTCGTGTTGTTGTTGAACAAGGTGTCGTTGCTCGTGAAATCGAAATGG GTGTACTTGGAAATGATACGCCAGTTTGCTCTGTACCTGGCGAAATTTTGCCAGAAGGTGCAGTTGCCAC ATTCTACGATTACAAAGCTAAATATCAAGATAACAATACTGCGTTAATTATTCCGACTGAAGTAGATCCA GAAATCTTGGAACAAATGAAAGAATATGCGATCCAAGCGTTTTTAGGTCTGGACGCAAGCGGACTAGTGC GAGCGGACTTTTTCTTAACGGAAGATAATCAGTTATTCTTAAATGAAGTGAATACAATGCCAGGTTTCAC ACCGTACAGCATGTATCCTCTTCTTTGGCAGGAAACGGGCTTACCGTACGGAGCGTTAATTGAACGATTA GTCGATTTAGCGAAAGAACGCCACGCAGCCAAAAATGCACTTAAATATAAATTAGAAGACTAA
 * Current Inhibitors **** : ** D-cycloserine
 * Expression Information **** (has it been expressed in bacterial cells): **http://www.ncbi.nlm.nih.gov/pubmed/1554356
 * Purification Method **** : ** Nickel affinity chromatography
 * Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence: **
 * *Length of your protein in Amino Acids: ** 370aa
 * Molecular Weight of your protein in kiloDaltons using the **[|**Expasy ProtParam**]** website: ** 40.6695 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 37360 M-1 cm-1
 * TMpred graph Image ** ( [] ). Input your amino acid sequence to it.
 * * **** CDS Gene Sequence: **
 * * **** GC% Content for gene: ** 39.712%


 * *CDS Gene Sequence (codon optimized): **

ATGAAAACCAAACTGATTCTGCTGTATGGTGGTAAATCTGCGGAACACGAAGTTTCCCTGCAGACCGCGTTCTCTGTTATCAATGCACTGGACCTCGAAAAATTCGAAGCGGCTCCGATCTACATCACCAACGAGGGTGAATGGATTCAGGGTCCGCTCCTGTCTGGTAAACTGGACTTCGTTGAACAGCTCCGTTTCTCTGCGACCGATACCATCAAACTGGCGACCACCGAATCTGAAAAGTCTGAAGGTGAAGCGATCTCTCCGGCGGTTCTGGAAGCGGACGGTCAAGAAACCGTTGTTTTCCCGCTGCTGCACGGTCCGAACGGTGAAGACGGTACTGTGCAGGGTCTGTTCGAAGTTCTGAACATCCCGTACGTTGGTAACGGTGTTCTGGCGTCCTCTGCGGCGATGGACAAAATCGTTATGAAGAAAATCTTCGCGGACGCGGGTATTCCGCAAGTACCGGCCGTTGCGGTTCGTCTGATCGACTGGAAGAATTACCAAGAGGAGATGGTTGCAGAAATGGAAGAGGTTCTGACCTACCCGGTCTTCGTTAAACCGGCGAATCTCGGTTCTTCTGTTGGTATCTCTAAAGCGACCAACAAAAAAGAACTGGTCGATGCGATGACTGAAGCGTTCCTGTACGACCGTCGCGTTGTTGTTGAGCAAGGTGTGGTTGCCCGTGAAATCGAGATGGGCGTACTGGGTAACGACACCCCGGTTTGCTCTGTACCGGGTGAGATCCTGCCGGAAGGTGCAGTTGCGACCTTCTATGACTACAAAGCGAAATACCAGGACAATAACACCGCCCTGATCATCCCTACCGAGGTTGACCCGGAAATCCTGGAACAGATGAAAGAGTACGCGATCCAGGCCTTCCTGGGTCTGGACGCCTCTGGTCTCGTTCGTGCGGACTTCTTCCTCACCGAAGACAACCAGCTCTTCCTGAATGAAGTTAACACCATGCCGGGTTTCACCCCGTATTCTATGTACCCACTCCTGTGGCAGGAGACCGGCCTGCCGTATGGCGCTCTGATTGAACGTCTGGTTGATCTGGCGAAAGAACGTCACGCGGCGAAAAACGCGCTGAAATACAAACTCGAAGACTAA


 * * **** GC% Content for gene (codon optimized): ** 51%

DNA Works output
 * Primer design results for pNIC-Bsa4 cloning: **

Forward Primer: 5'-TACTTCCAATCCATGAAAACCAAACTGATTCTG-3' Reverse Primer: 5'-TATCCACCTTTACTGTTAGTCTTCGAGTTTGTATTTCA-3'
 * Primer design results for 'tail' primers (this is just 2 sequences): **