D-alanine+Ligase.+(M.+tuberculosis)

http://www.absa.org/riskgroups/bacteriasearch.php?genus=Mycobacterium&species=tuberculosis
 * Target (protein/gene name):** D-alanine:D-alanine Ligase
 * NCBI Gene # or RefSeq#:** Rv2981c (Tuberculist ID)
 * Protein ID (NP or XP #) or Wolbachia#:** //N/A//
 * Organism (including strain):** Mycobacterium tuberculosis
 * Etiologic Risk Group (see link below):** 3


 * Background/ Disease Information (sort of like the Intro to your Mini __Research Write__ up):**

//Mycobacterium tuberculosis// is the leading bacteria that causes the pathogenic disease tuberculosis. This infectious disease, which is often times shortened to TB, is commonly spread through the air and infects various parts of the body, mainly the lungs. The common symptoms of TB are: a bad cough that lasts more than three weeks, chest pains, coughing of blood, fatigue, weight loss, chills, fever, sweating at night, and loss of appetite. Most tuberculosis diagnosis are made through chest x-rays and microbiological cultures of bodily fluids. Tuberculosis is the second most common cause of death from infectious diseases. Due to lowered immune systems, it is highly present in areas of malnutrition and poverty along with those who have or at risk of having HIV. Many Asian and African countries are largely exposed to tuberculosis. Tuberculosis is not spread by physical contact like shaking someone’s hand, sharing food and drinks, kissing, etc. There are two types of tuberculosis related conditions: latent TB infection and TB disease. Latent TB infection is where an individual is not sick but is still infected with // Mycobacterium tuberculosis //. The reason why the individual is not sick but still infected is due to their immune system which is preventing the growth of // Mycobacterium tuberculosis // and stops the spread of the bacteria to the environment. If the // Mycobacterium tuberculosis // does grow, then the infection will proceed to the actual tuberculosis disease where the affected individual can now infect others. The reason for the bacteria growing may be a result of an HIV infection, recently infected with TB bacteria, alcohol or drug abuse, or other health problems. The treatment for latent TB infection consists of medicine called isoniazid (INH),rifampin (RIF), rifapentine (RPT). These medicines prevent // Mycobacterium tuberculosis // from growing and becoming active. The treatment for TB disease consists of an individual taking approximately seven drugs for 6 to 9 months. The main medicine that the individual must take include:isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA).

http://tdrtargets.org/targets/view?gene_id=5855
 * Link to TDR Targets page (if present):**

Is it a monomer or multimer as biological unit**? (make prediction at** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): 0.5 http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.2.4
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)**
 * Essentiality of this protein:** mixed essentiality
 * Complex of proteins?:** No, because chains A and B are both the same which means that it is a dimer.
 * 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:**

Figure 1: Reaction diagram of D-alanine:D-alanine ligase from BRENDA site.

Figure 2: PyMol images of D-alanine and ADP attached to Ddl. ADP is indicated by the enzyme with atoms colored in yellow; D-alanine is the enzyme shown in red. http://aac.asm.org/content/55/1/291.figures-only

http://aac.asm.org/content/55/1/291.long
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**


 * -- 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 #**

Use non-radioactive assay; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149019/ malachite green assay

PDB #: **3LWB**
 * Structure (PDB or Homology model)**

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.2.4
 * Current Inhibitors:**

Competitively inhibited by: D-cycloserine - "preferential and weak inhibition at the second, lower-affinity binding site. D-cycloserine binding is tighter at higher ATP concentrations"

Escherichia coli
 * Expression Information (has it been expressed in bacterial cells):**

Reading literature. Pending.
 * Purification Method: **


 * Image of protein (PyMol with features delineated and shown separately):**

PyMol images of 3LWB (from left to right) shown in sticks with chains A and B shown in blue and green, and another image with the backbone in cartoon,

http://tuberculist.epfl.ch/quicksearch.php?gene+name=Rv2981c
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

MSANDRRDRRVRVAVVFGGRSNEHAISCVSAGSILRNLDSRRFDVIAVGITPAGSWVLTD ANPDALTITNRELPQVKSGSGTELALPADPRRGGQLVSLPPGAGEVLESVDVVFPVLHGP YGEDGTIQGLLELAGVPYVGAGVLASAVGMDKEFTKKLLAADGLPVGAYAVLRPPRSTLH RQECERLGLPVFVKPARGGSSIGVSRVSSWDQLPAAVARARRHDPKVIVEAAISGRELEC GVLEMPDGTLEASTLGEIRVAGVRGREDSFYDFATKYLDDAAELDVPAKVDDQVAEAIRQ LAIRAFAAIDCRGLARVDFFLTDDGPVINEINTMPGFTTISMYPRMWAASGVDYPTLLAT MIETTLARGVGLH //*The beginning amino acid was switched from V to M.//
 * length of your protein in Amino Acids = 373**

39.710 kDa, or 39710.2 Da
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:**

code Ext. coefficient   27180 Abs 0.1% (=1 g/l)  0.684, assuming all pairs of Cys residues form cystines
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**

Ext. coefficient   26930 Abs 0.1% (=1 g/l)  0.678, assuming all Cys residues are reduced code


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



gtgagtgctaacgaccggcgtgatcggcgtgtccgcgttgccgtcgtgttcggcgggcgcagcaacgagcacgccatctcgtgtgtgtccgccggcagcatcctgcgcaacctggactcg cggcggttcgacgtgatcgcggtgggtatcaccccggcaggttcgtgggtgctcaccgacgccaaccccgacgccctgacgatcaccaaccgggagcttcctcaggtcaaatcaggatcg ggcaccgagctggcgctgccggccgatccgcggcgtggtggccagttggtgtcgctgccgcccggggccggcgaggttctggagtcggtcgacgtggtgttcccggtactgcacggcccgt acggcgaggacggcacgatccagggactgctcgaactcgccggggtgccctacgtgggcgccggtgtgctggccagtgccgtcggcatggacaaggagttcaccaagaagctgctcgc cgccgatggacttccggtgggtgcgtacgcggtgctgcgtccgccgcggtcgacactgcaccgccaggagtgcgaacggctgggcttaccggtgttcgtcaaacccgcccgaggcggctc gtcgatcggtgttagccgggtgtcgagttgggatcaactgcccgccgcggtcgcgcgggcccgccggcatgaccctaaggtcatcgtcgaggccgcgatcagcggccgcgagctggaatgc ggtgtgctcgaaatgccggacggcacactggaagccagcacgctgggggagatccgggtggccggggtgcggggacgcgaggactctttctacgacttcgcaaccaagtatctcgacgac gcagccgaattggacgtgcccgccaaggtcgatgaccaggtcgcagaggcgattcgtcagctggcgatccgggcgttcgcggctatcgactgccggggtctggccagggtggacttcttcctc accgacgacggtccggtgatcaacgagatcaacacgatgccgggattcaccacgatctcgatgtacccgcggatgtgggcggccagcggtgtcgactatccgaccctgctggcgacgatg atcgagacgacattggcccgcggcgtgggcctgcactag
 * CDS Gene Sequence (paste as text only): from Tuberculist**


 * GC% Content for gene:** 67.47%

==== ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCGCGTCGCTGTTGTGTTCGGTGGCCGCTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTCT CGCCGTTTCGACGTTATCGCGGTAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCATTACTAACCGTGAACTCCCGCAGGTAAAATCTGGTTCT GGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCAACTCGTTTCTCTCCCACCGGGTGCGGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCG TATGGTGAAGATGGTACGATCCAGGGCCTGCTGGAGCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAAAGAATTCACCAAAAAACTGCTGGCA GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCATCGTCAAGAATGCGAGCGTCTGGGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCT TCTATTGGTGTTTCTCGTGTTTCCTCTTGGGATCAGCTCCCTGCGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTGGTCGTGAGCTGGAATGT GGTGTCCTGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCGTGCGTGGCCGTGAGGACTCTTTCTACGACTTTGCGACCAAATATCTGGACGAC GCAGCCGAACTGGATGTTCCGGCGAAGGTTGATGATCAGGTGGCTGAAGCTATCCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGACTTCTTC CTGACTGATGATGGTCCGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTATGTGGGCGGCATCTGGCGTTGATTACCCGACCCTGCTCGCGACC ATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCATTAA ====
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized):** 57.7%

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. ** DNA sequence for Mycobacterium Tuberculosis from DNA Works Ouput. ** ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCGCGTCGCTGTTGTGTTCGGTGGCCGCTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTCT CGCCGTTTCGACGTTATCGCGGTAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCATTACTAACCGTGAACTCCCGCAGGTAAAATCTGGTTCT GGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCAACTCGTTTCTCTCCCACCGGGTGCGGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCG TATGGTGAAGATGGTACGATCCAGGGCCTGCTGGAGCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAAAGAATTCACCAAAAAACTGCTGGCA GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCATCGTCAAGAATGCGAGCGTCTGGGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCT TCTATTGGTGTTTCTCGTGTTTCCTCTTGGGATCAGCTCCCTGCGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTGGTCGTGAGCTGGAATGT GGTGTCCTGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCGTGCGTGGCCGTGAGGACTCTTTCTACGACTTTGCGACCAAATATCTGGACGAC GCAGCCGAACTGGATGTTCCGGCGAAGGTTGATGATCAGGTGGCTGAAGCTATCCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGACTTCTTC CTGACTGATGATGGTCCGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTATGTGGGCGGCATCTGGCGTTGATTACCCGACCCTGCTCGCGACC ATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCATTAA
 * 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)
 * B12 || MtDdla_1 || ATGTCTGCTAACGACCGCCGTGACCGTCGCGTTCG ||
 * C1 || MtDdla_2 || AGATGGCGTGTTCGTTAGAGCGGCCACCGAACACAACAGCGACGCGAACGCGACGGTCAC ||
 * C2 || MtDdla_3 || CTCTAACGAACACGCCATCTCTTGCGTTTCTGCGGGTTCTATCCTGCGTAATCTCGACTC ||
 * C3 || MtDdla_4 || GCCGGGGTGATGCCTACCGCGATAACGTCGAAACGGCGAGAGTCGAGATTACGCAGGATA ||
 * C4 || MtDdla_5 || TAGGCATCACCCCGGCAGGCTCCTGGGTTCTGACCGATGCGAACCCGGACGCACTGACCA ||
 * C5 || MtDdla_6 || CGTACCAGAACCAGATTTTACCTGCGGGAGTTCACGGTTAGTAATGGTCAGTGCGTCCGG ||
 * C6 || MtDdla_7 || GGTAAAATCTGGTTCTGGTACGGAGCTGGCACTGCCTGCGGACCCTCGTCGTGGTGGCCA ||
 * C7 || MtDdla_8 || CAGATTCCAGAACTTCGCCCGCACCCGGTGGGAGAGAAACGAGTTGGCCACCACGACGAG ||
 * C8 || MtDdla_9 || GGGCGAAGTTCTGGAATCTGTTGACGTTGTTTTCCCAGTTCTGCACGGCCCGTATGGTGA ||
 * C9 || MtDdla_10 || AGGTACACCCGCGAGCTCCAGCAGGCCCTGGATCGTACCATCTTCACCATACGGGCCGTG ||
 * C10 || MtDdla_11 || GCTCGCGGGTGTACCTTATGTTGGTGCAGGTGTTCTCGCGTCTGCGGTGGGTATGGACAA ||
 * C11 || MtDdla_12 || ACTGGGAGGCCATCTGCTGCCAGCAGTTTTTTGGTGAATTCTTTGTCCATACCCACCGCA ||
 * C12 || MtDdla_13 || GCAGATGGCCTCCCAGTAGGTGCCTACGCGGTCCTCCGTCCGCCTCGTTCTACCCTCCAT ||
 * D1 || MtDdla_14 || TTAACGAAAACCGGCAGACCCAGACGCTCGCATTCTTGACGATGGAGGGTAGAACGAGGC ||
 * D2 || MtDdla_15 || GGTCTGCCGGTTTTCGTTAAACCTGCCCGTGGCGGCTCTTCTATTGGTGTTTCTCGTGTT ||
 * D3 || MtDdla_16 || CACGCGCAACTGCCGCAGGGAGCTGATCCCAAGAGGAAACACGAGAAACACCAATAGAAG ||
 * D4 || MtDdla_17 || CGGCAGTTGCGCGTGCCCGTCGCCATGACCCGAAAGTTATCGTTGAAGCGGCGATCTCTG ||
 * D5 || MtDdla_18 || ACCGTCCGGCATTTCCAGGACACCACATTCCAGCTCACGACCAGAGATCGCCGCTTCAAC ||
 * D6 || MtDdla_19 || TGGAAATGCCGGACGGTACCCTCGAAGCGTCTACTCTGGGCGAAATTCGTGTGGCAGGCG ||
 * D7 || MtDdla_20 || TATTTGGTCGCAAAGTCGTAGAAAGAGTCCTCACGGCCACGCACGCCTGCCACACGAATT ||
 * D8 || MtDdla_21 || TCTACGACTTTGCGACCAAATATCTGGACGACGCAGCCGAACTGGATGTTCCGGCGAAGG ||
 * D9 || MtDdla_22 || CGAATTGCGAGCTGACGGATAGCTTCAGCCACCTGATCATCAACCTTCGCCGGAACATCC ||
 * D10 || MtDdla_23 || CCGTCAGCTCGCAATTCGCGCATTCGCTGCGATTGACTGCCGTGGCCTCGCACGTGTGGA ||
 * D11 || MtDdla_24 || TGTTGATCTCGTTGATCACCGGACCATCATCAGTCAGGAAGAAGTCCACACGTGCGAGGC ||
 * D12 || MtDdla_25 || CGGTGATCAACGAGATCAACACTATGCCGGGCTTTACTACCATTTCTATGTACCCGCGTA ||
 * E1 || MtDdla_26 || CGAGCAGGGTCGGGTAATCAACGCCAGATGCCGCCCACATACGCGGGTACATAGAAATGG ||
 * E2 || MtDdla_27 || TTACCCGACCCTGCTCGCGACCATGATCGAAACCACCCTGGCTCGTGGCGTCGGTCTCCA ||
 * E3 || MtDdla_28 || TTAATGGAGACCGACGCCAC ||


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

TACTTCCAATCCATG __ TCTGCTAACGACCGCC __
 * Forward Primer:**

TATCCACCTTTACTGTTA__ATGGAGACCGACGCCAC__
 * Reverse Complement Primer:**

NCBI LINKS ** http://www.ncbi.nlm.nih.gov/gene/888415 - ddlA D-alanine--D-alanine ligase [ //Mycobacterium tuberculosis H37Rv// ] http://www.ncbi.nlm.nih.gov/gene/13317778 - RVBD_2981c d-alanine ligase DdlA [ //Mycobacterium tuberculosis H37Rv// ]