D-alanine+Ligase+(Staphylococcus+aureus)


 * *Target (protein/gene name): **D-alanine: D-alanine Ligase


 * Prefix:** SaDAL


 * *NCBI Gene # or RefSeq#: **23197880


 * *Protein ID (NP or XP #) or Wolbachia#: **KEK52203.1


 * *Organism (including strain): **Staphylococcus aureus


 * Etiologic Risk Group (see link below): **

// Staphylococcus // //aureus// is a gram positive bacterium commonly found among the human body. It is a common cause of several infections, and is increasingly growing resistant to antibiotics. Methicillin resistant //Staphylococcus aureus// (MRSA) is the leading cause of nosocomial, or hospital-acquired, pneumonia and is the second leading cause of nosocomial blood stream infections. MRSA is becoming more prevalent within the United States, and as a result, an increasing demand for novel antibiotics has emerged.
 * */ Disease Information (sort of like the Intro to your Mini __Research Write__ up): **


 * Link to TDR Targets page (if present): ** not available

NCBI - http://www.ncbi.nlm.nih.gov/gene/?term=23197880 UniProt - http://www.uniprot.org/uniprot/Q2FWH3#entry_information PATRIC - https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.985762.7.JPRT010 00013.CDS.45731.46801.fwd
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **

D-alanine ligase is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. Mutations in the protein has caused resistance against the antibiotic that's used as last resort, D-Alanine Metabolism plays a role in the metabolism of other amino acids. D-Alanine ligase is an essential enzyme in bacterial cell wall biosynthesis.
 * Essentiality of this protein: **

Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Most likely a 2-mer (dimer)


 * Complex of proteins?: **dimer


 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **


 * *EC#: **6.3.2.4

http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.4
 * Link to BRENDA EC# page: **

Figure 1. Reaction catalyzed by D-alanine: D-alanine ligase. (EC 6.3.2.4)


 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): ** Malachite green phosphate assay

https://www.caymanchem.com/pdfs/10009325.pdf
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **

AquaPhoenix ATP (2g), Fisher-Scientific, Catalog No. S25123…………………..$54.00 https://www.fishersci.com/shop/products/aquaphoenix-adenosine-triphosphate-atp/s25123 D-Alanine, +99% (5g), Fisher-Scientific, Catalog No. AC172080050…………….$30.00 https://www.fishersci.com/shop/products/d-alanine-99-acros-organics-3/p-4259097
 * -- links to assay reagents (substrates) pages. **
 * --- List cost and quantity of substrate reagents, supplier, and catalog no. **

4 structures available: 3N8D, 2I80, 2I8C, 2I87 Structures for Screening: 3N8D (Anthony) ???? (Kevin)
 * Structure (PDB): **

vancomycin (developed resistance); Bruton's tyrosine kinase inhibitor LFM-A13
 * Current Inhibitors: **

Successfully expressed in // E. coli //M15 (pREP4) http://www.pnas.org/content/103/41/15178
 * Expression Information (has it been expressed in bacterial cells): **


 * Purification Method : **Nickel affinity chromatography

Figure 2. Crystal structure of D-alanine: D-alanine Ligase from //Staphylococcus aureus//,shown asPyMOL representation.Protein chains shown as cartoon and sticks colored separately on spectrum gradient. (PDB: 3N8D)
 * Image of protein (PyMol with features delineated and shown separately): **

mtkenicivfggksaehevsiltaqnvlnaidkdkyhvdiiyitndgdwrkqnnitaeik stdelhlengealeisqllkesssgqpydavfpllhgpngedgtiqglfevldvpyvgng vlsaassmdklvmkqlfehrglpqlpyisflrseyekyehnilklvndklnypvfvkpan lgssvgiskcnneaelkegikeafqfdrklvieqgvnareievavlgndypeatwpgevv kdvafydykskykdgkvqlqipadldedvqltlrnmaleafkatdcsglvradffvtedn qiyinetnampgftafsmypklwenmglsypelitklielakerhqdkqknkykid
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **


 * *length of your protein in Amino Acids: **356

40 kDa (40230.5 Da)
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website **

40465 M^-1 cm^-1 (assuming all pairs of Cys residues form cystines) 40340 M^-1 cm^-1 (assuming all Cys residues are reduced)
 * Molar Extinction coefficient of your protein at 280 nm wavelength: **


 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

ATGACAAAAGAAAATATTTGTATCGTTTTTGGAGGGAAAAGTGCAGAACACGAAGTATCG ATTCTGACAGCACAAAATGTATTAAATGCAATAGATAAAGACAAATATCATGTTGATATC ATTTATATTACCAATGATGGTGATTGGAGAAAGCAAAATAATATTACAGCTGAAATTAAA TCTACTGATGAGCTTCATTTAGAAAATGGAGAGGCGCTTGAGATTTCACAGCTATTGAAA GAAAGTAGTTCAGGACAACCATACGATGCAGTATTCCCATTATTACATGGTCCTAATGGT GAAGATGGCACGATTCAAGGGCTTTTTGAAGTTTTGGATGTACCATATGTAGGAAATGGT GTATTGTCAGCTGCAAGTTCTATGGACAAACTTGTAATGAAACAATTATTTGAACATCGA GGGTTACCACAGTTACCTTATATTAGTTTCTTACGTTCTGAATATGAAAAATATGAACAT AACATTTTAAAATTAGTAAATGATAAATTAAATTACCCAGTCTTTGTTAAACCTGCTAAC TTAGGGTCAAGTGTAGGTATCAGTAAATGTAATAATGAAGCGGAACTTAAAGAAGGTATT AAAGAAGCATTCCAATTTGACCGTAAGCTTGTTATAGAACAAGGCGTTAACGCACGTGAA ATTGAAGTAGCAGTTTTAGGAAATGACTATCCTGAAGCGACATGGCCAGGTGAAGTCGTA AAAGATGTCGCGTTTTACGATTACAAATCAAAATATAAAGATGGTAAGGTTCAATTACAA ATTCCAGCTGACTTAGACGAAGATGTTCAATTAACGCTTAGAAATATGGCATTAGAGGCA TTCAAAGCGACAGATTGTTCTGGTTTAGTCCGTGCTGATTTCTTTGTAACAGAAGACAAC CAAATATATATTAATGAAACAAATGCAATGCCTGGATTTACGGCTTTCAGTATGTATCCA AAGTTATGGGAAAATATGGGCTTATCTTATCCAGAATTGATTACAAAACTTATCGAGCTT GCTAAAGAACGTCACCAGGATAAACAGAAAAATAAATACAAAATTGACTAA
 * *CDS Gene Sequence (paste as text only): **


 * *GC% Content for gene: ** 33.8

ATGACCAAAGAAAATATCTGCATCGTTTTCGGTGGTAAGTCTGCGGAACACGAAGTTTCT ATTCTGACCGCGCAGAATGTTCTCAACGCGATCGACAAAGATAAATACCACGTTGACATC ATCTATATTACCAATGACGGTGACTGGCGTAAACAAAACAATATCACGGCGGAAATCAAG AGCACGGACGAGCTGCATCTGGAAAATGGTGAGGCGCTGGAAATCTCTCAGCTCCTGAAA GAATCTTCTAGCGGTCAACCGTACGACGCAGTTTTTCCGCTGCTGCATGGCCCGAATGGC GAGGATGGTACTATCCAAGGTCTGTTCGAAGTTCTGGACGTTCCTTATGTTGGTAACGGT GTGCTGTCTGCAGCGTCTTCTATGGATAAACTCGTCATGAAGCAGCTGTTTGAACACCGT GGTCTGCCGCAGCTCCCGTACATCTCTTTCCTGCGTTCTGAATATGAAAAATACGAACAC AACATCCTGAAGCTGGTGAACGACAAACTGAACTATCCTGTTTTTGTTAAGCCAGCGAAT CTGGGCAGCTCTGTTGGCATCTCTAAATGCAACAACGAAGCCGAACTCAAAGAAGGTATC AAGGAAGCGTTCCAGTTTGACCGTAAACTGGTAATTGAACAGGGTGTTAATGCGCGCGAG ATCGAGGTGGCTGTCCTGGGCAATGATTACCCAGAAGCAACCTGGCCGGGTGAAGTAGTT AAAGACGTCGCGTTTTATGACTATAAGTCTAAATACAAGGACGGTAAAGTTCAACTCCAG ATCCCAGCGGACCTGGACGAAGATGTTCAGCTCACCCTGCGCAACATGGCACTGGAAGCC TTCAAAGCGACCGACTGCTCTGGTCTGGTTCGTGCGGACTTTTTCGTCACCGAGGACAAC CAGATTTACATCAATGAGACCAATGCGATGCCGGGTTTCACTGCATTTTCTATGTACCCG AAACTGTGGGAGAATATGGGTCTGAGCTATCCGGAACTCATCACTAAACTGATCGAACTG GCGAAAGAGCGTCACCAGGATAAGCAGAAGAATAAGTACAAAATTGACTAA
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **


 * *GC% Content for gene (codon optimized): ** 47.6