Target (protein/gene name): UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase (Rv2158c)
NCBI Gene # or RefSeq#: NC_000962.3
Protein ID (NP or XP #) or Wolbachia#: NP_216674.1
Organism (including strain): Mycobacterium tuberculosis (H37Rv)
Etiologic Risk Group (see link below): N/A
Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium tuberculosis is a pathogenic-type bacteria that is in charge of the infectious Tuberculosis disease. Tuberculosis is highly infective, known for having a high morbidity and a high mortality rate. The disease starting infecting more and becoming more severe after 1985 when HIV entered into body systems and lowered the immunity of the infected. The severity of the bacteria is contagious by its microscopic bodily fluids
out in the air that can infect the surface in the lungs which secretes pulmonary surfactant that decreases the surface tension of the alveoli. The most common symptoms in a Tuberculosis-infected patient are coughing, fatigue, a high fever, and chest pain. Those who have poor nutritional diets, live in places that do not offer medical care, or are highly populated risk getting Tuberculosis. Because of its resiliency tendency, M.tuberculosis bacterium undergo quick mutations resulting in more resistant strains compared to other bacteria, so it is important to keep up with the strains and research in order to prevent the spread of the disease.

Link to TDR Targets page (if present):
http://www.tdrtargets.org/targets/view?gene_id=6551
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.patricbrc.org/view/Feature/PATRIC.1643430.3.LDXW01000001.CDS.103333.104823.rev
https://www.ncbi.nlm.nih.gov/gene/?term=2.4.2.8+mycobacterium+tuberculosis

Essentiality of this protein:
Rv2158c has essentiality data

Gene/Ortholog: eco86 (OG4_14754); Phenotype: undefined; Source study: blattner

Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: gerdes

Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: keio

Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: shigen


Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
No it is not.

EC#:
**6.3.2.13**

Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.13&UniProtAcc=P9WJL3&OrganismID=3445

Link to BRENDA HPTR Pathway:
http://www.brenda-enzymes.org/pathway_index.php?menu=off&site=pathway&search_mode=exact&pathway=peptidoglycan_biosynthesis&ecno=6.3.2.13

Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
colorimetric phosphate detection method
[1] https://www.researchgate.net/publication/45707755_Anti-tubercular_screening_of_natural_product_from_Colombian_plants_3-methoxynordomesticine_an_inhibitor_of_MurE_ligase_of_Mycobacterium_tuberculosis
[2] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498294/

Fluorogenic substrates, DMSO and PBS
DMSO - $68.50 for 100 mL
PBS - $110 for 1L

Structure (PDB or Homology model)
PDB # or closest PDB entry if using homology model:
2WTZ
http://www.rcsb.org/structure/2WTZ

Current Inhibitors:
Nine compounds for the Target in M. tuberculosis

Expression Information (has it been expressed in bacterial cells):
[1] Chandrakala Basavannacharya, Giles Robertson, Tulika Munshi, Nicholas H. Keep, Sanjib Bhakta Tuberculosis (Edinb) 2010 Jan; 90(1): 16–24. Published online 2009 Nov 27. doi: 10.1016/j.tube.2009.10.007 ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.
[2] Basavannacharya C, Moody PR, Munshi T, Cronin N, Keep NH, Bhakta S. Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis. Protein & Cell. 2010;1(11):1011-1022. doi:10.1007/s13238-010-0132-9.

Image of protein (PyMol with features delineated and shown separately):
Picture1.pngprotein pic.png
Fig 1. Crystal structure of Mycobacterium tuberculosis UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase compared with the 2WTZ MurE ligase

Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>2WTZ:A|PDBID|CHAIN|SEQUENCE
MSSLARGISRRRTEVATQVEAAPTGLRPNAVVGVRLAALADQVGAALAEGPAQRAVTEDRTVTGVTLRAQDVSPGDLFAA
LTGSTTHGARHVGDAIARGAVAVLTDPAGVAEIAGRAAVPVLVHPAPRGVLGGLAATVYGHPSERLTVIGITGTSGKTTT
TYLVEAGLRAAGRVAGLIGTIGIRVGGADLPSALTTPEAPTLQAMLAAMVERGVDTVVMEVSSHALALGRVDGTRFAVGA
FTNLSRDHLDFHPSMADYFEAKASLFDPDSALRARTAVVCIDDDAGRAMAARAADAITVSAADRPAHWRATDVAPTDAGG
QQFTAIDPAGVGHHIGIRLPGRYNVANCLVALAILDTVGVSPEQAVPGLREIRVPGRLEQIDRGQGFLALVDYAHKPEAL
RSVLTTLAHPDRRLAVVFGAGGDRDPGKRAPMGRIAAQLADLVVVTDDNPRDEDPTAIRREILAGAAEVGGDAQVVEIAD
RRDAIRHAVAWARPGDVVLIAGKGHETGQRGGGRVRPFDDRVELAAALEALERRA

length of your protein in Amino Acids
535 AA
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
55.34076 kDa
Molar Extinction efficient of your protein at 280 nm wavelenth:
18575

TMpred graph Image TMPRED
Input your amino acid sequence to it.
Picture3.png

Fig. 2 TMpred output for UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopi melate-D-alanyl-D-alanyl ligase
**Gene Sequence (paste as text only):**
>>Mycobacterium tuberculosis H37Rv|CDS|Rv2158c|2419001-2420608|-|murE|downstream:0|upstream:0
gtgtcatcgctggcccgagggatctcgcggcggcgaacggaggtggcgacacaggtggaggctgcgcccactg
gcttgcgccccaacgccgtcgtgggcgttcggttggccgcactggccgatcaggtcggcgcggccctggccga
gggtccagctcagcgtgccgtcaccgaggaccggacggtcaccggggtcacgctgcgcgcccaggacgtgtca
cccggtgacctgttcgccgccctgaccggctcgaccacccacggggcccgccacgtcggcgacgcgatcgcac
gcggcgccgtcgcggtgctcaccgaccccgccggggtcgccgagatcgccggacgagcggccgtgcccgtgtt
ggtgcaccccgcaccccgcggcgtgctcggcggcttggccgccaccgtgtacgggcatccgtccgagcggttg
acggttatcgggatcaccggaacgtccggcaagaccaccaccacctatctggtcgaggccgggttacgggctg
ccggacgcgtcgccgggctgatcggcaccatcggcatccgcgtcggcggcgccgaccttcccagcgcgctgac
caccccggaggcccccacgctgcaggcgatgctggcggcgatggtcgaacgcggggtggacaccgtggtcatg
gaggtgtccagccacgcgctggcgctgggccgggtggacggcacccggttcgccgtcggcgccttcaccaatc
tctcccgtgaccacctggatttccaccccagcatggccgactacttcgaggccaaggcgtcattgttcgatcc
ggactcggcactgcgcgcccgcaccgccgtggtgtgcatcgacgacgacgccgggcgcgcgatggcggcgcgg
gccgccgacgcgatcaccgtcagcgccgccgaccggcccgcacactggcgcgccacggatgtggcgcccacgg
acgcgggcgggcaacaattcaccgccatcgaccccgccggcgtagggcatcacatcggaatccggctaccggg
ccgctacaacgtcgccaattgcctggtcgccctggcgattctggacaccgtcggggtctccccggaacaggcg
gtgccgggcctgcgtgagatccgggtcccggggcggctcgagcagatcgaccgcggccagggctttctcgcgc
tggtcgactacgcgcacaaaccggaagcgctgcggtcggtgctgaccaccttggcgcacccggaccgccggct
ggcggtggtgttcggcgccggcggcgatcgtgacccgggcaagcgggccccgatgggccggatagccgcgcag
ctggccgacttggtggtcgtcaccgacgacaacccgcgtgacgaagatcccacggcgatccgccgcgaaatcc
tggctggggcggccgaagtcggcggtgatgcccaggtcgtcgagatcgcagaccggcgggacgcgatccggca
cgcggttgcctgggcgcgccccggcgacgtggtgctcatcgccggcaaaggccacgagaccgggcaacgcggc
ggcgggcgggtccgcccgttcgacgaccgggtggagctggctgccgcgctagaggccctcgagcggcgcgcatga
**Protein Sequence (paste as text only):**
>>Mycobacterium tuberculosis H37Rv|Rv2158c|murE
        10         20         30         40         50
MSSLARGISR RRTEVATQVE AAPTGLRPNA VVGVRLAALA DQVGAALAEG
        60         70         80         90        100
PAQRAVTEDR TVTGVTLRAQ DVSPGDLFAA LTGSTTHGAR HVGDAIARGA
       110        120        130        140        150
VAVLTDPAGV AEIAGRAAVP VLVHPAPRGV LGGLAATVYG HPSERLTVIG
       160        170        180        190        200
ITGTSGKTTT TYLVEAGLRA AGRVAGLIGT IGIRVGGADL PSALTTPEAP
       210        220        230        240        250
TLQAMLAAMV ERGVDTVVME VSSHALALGR VDGTRFAVGA FTNLSRDHLD
       260        270        280        290        300
FHPSMADYFE AKASLFDPDS ALRARTAVVC IDDDAGRAMA ARAADAITVS
       310        320        330        340        350
AADRPAHWRA TDVAPTDAGG QQFTAIDPAG VGHHIGIRLP GRYNVANCLV
       360        370        380        390        400
ALAILDTVGV SPEQAVPGLR EIRVPGRLEQ IDRGQGFLAL VDYAHKPEAL
       410        420        430        440        450
RSVLTTLAHP DRRLAVVFGA GGDRDPGKRA PMGRIAAQLA DLVVVTDDNP
       460        470        480        490        500
RDEDPTAIRR EILAGAAEVG GDAQVVEIAD RRDAIRHAVA WARPGDVVLI
       510        520        530
AGKGHETGQR GGGRVRPFDD RVELAAALEA LERRA
Do Not Need this info for Spring (but still copy these lines to your Target page for now
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers
link to DNA Works output m9l40l772 that should be saved in your folder after you did the primer design protocol
-- 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 'tail' primers (this is just 2 sequences):