Target+-+β-carbonic+anhydrases+(Mycobacterium+tuberculosis)

(http://tdrtargets.org/targets/view?gene_id=7856)
 * Target (protein/gene name):**β-carbonic anhydrases
 * NCBI Gene # or RefSeq#:** GI:78101214

Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
 * Protein ID:** 1YLK
 * Organism (including strain):** Mycobacterium tuberculosis H37Rv
 * Etiologic Risk Group (see link below):**

Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Three β- carbonic anhydrases are present in the human pathogen Mycobacterium tuberculosis. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta- carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta- carbonic anhydrase can switch between two states. There is a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.
 * Background/Disease Information: **
 * Essentiality of this protein: **
 * Gene/Ortholog:** mtu1305 (OG4_19505); **Phenotype:** essential; **Source study:** nmpdr


 * Complex of proteins?:** No
 * Druggable Target:** It's known to be druggable by orthology.

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.1.1
 * EC#: ** 4.2.1.1
 * Link to BRENDA EC# page:**


 * Screenshot of BRENDA enzyme mechanism schematic: **

http://www.sigmaaldrich.com/img/assets/18220/Carbonic_Anhydrase_-_CSRD.pdf http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/n8130pis.pdf
 * Enzyme Assay information:**
 * -- link to assay or assay reagents (substrates):**

Though enzyme activity can be measured by CO2 hydration, the surrogate enzyme assay using 4-nitrophenyl acetate, measuring the amount of 4-nitrophenol cleaved.

http://pubs.acs.org/doi/pdf/10.1021/jm9000488
 * -- link (or citation) to paper that contains assay information**


 * -- Cost and quantity of substrate reagents and supplier:** N8130-10G $48.20

-- PDB # or closest PDB entry if using homology model: 1YLK -- For Homology Model option: methanoarchaen Methanobacterium thermoautotrophicum (Cab). Show pairwise alignment of your BLASTP search in NCBI against the PDB Query Coverage: % Positives Max % Identities: Chain used for homology: Figure 1: PyMol representation of the protein target, β-carbonic anhydrases, displayed as cartoon with the ligand, SCN shown as sticks. 2-amino-pyrimidin-4-yl-sulfanilamide, 2-aminobenzenesulfonamide, 3-(4-sulfamoylphenyl)propanoic acid, 3-bromosulfanilamide, 4,5-dichloro-benzene-1,3-disulfonamide.
 * Structure Available (PDB or Homology model)**
 * Current Inhibitors:**

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.1.1&Suchword=&organism%5B%5D=Mycobacterium+tuberculosis&show_tm=0

expressed in Escherichia coli glutathione Sepharose 4B column chromatography and sulfonamide affinity chromatography
 * Expression Information (has it been expressed in bacterial cells):**
 * Purification Method:**
 * Image of protein (PyMol with features delineated and shown separately):**

ATGACCGTTACCGATGACTACCTGGCGAATAACGTTGACTACGCGTCTGGTTTCAAAGGT CCGCTGCCGATGCCGCCGTCTAAACACATCGCAATCGTTGCGTGCATGGACGCACGTCTG GACGTTTACCGCATGCTGGGTATCAAAGAAGGTGAAGCGCATGTCATCCGCAACGCGGGT TGCGTTGTAACCGACGACGTTATCCGTTCTCTGGCGATCTCTCAGCGTCTGCTCGGTACC CGTGAGATCATCCTGCTGCACCACACCGACTGCGGTATGCTGACCTTCACGGACGACGAC TTCAAACGTGCCATCCAGGACGAAACTGGTATCCGTCCGACCTGGTCTCCGGAATCTTAC CCGGACGCGGTTGAAGACGTTCGTCAGAGCCTGCGCCGTATCGAAGTTAACCCGTTCGTT ACCAAACACACCTCTCTGCGTGGTTTCGTTTTCGACGTTGCGACCGGCAAACTGAACGAG GTTACCCCGTAA
 * Amino Acid Sequence:**


 * Length of protein:** 172 amino acids; 516 base pairs
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website**

code 22540.4 code

code Ext. coefficient   10095 Abs 0.1% (=1 g/l)  0.448, assuming all pairs of Cys residues form cystines
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**

Ext. coefficient    9970 Abs 0.1% (=1 g/l)  0.442, assuming all Cys residues are reduced code code >gi|448814763:1437324-1437815 Mycobacterium tuberculosis H37Rv complete genome GTGACGGTTACCGACGACTACCTGGCCAACAACGTGGACTACGCGAGCGGTTTCAAGGGCCCGCTACCGA TGCCGCCGAGCAAACACATCGCAATCGTGGCGTGCATGGACGCCCGGCTGGACGTCTACCGCATGCTGGG CATCAAGGAGGGCGAGGCACACGTCATCCGCAACGCCGGATGCGTGGTCACCGACGATGTGATCCGTTCA CTGGCCATCAGCCAGCGGCTGCTGGGAACCCGCGAAATCATCCTGCTGCACCACACCGACTGTGGGATGC TGACTTTCACCGACGACGACTTCAAGCGCGCCATCCAGGACGAGACCGGCATCAGACCCACGTGGTCGCC CGAGTCGTACCCCGACGCCGTCGAGGACGTCCGTCAGTCGCTGCGCCGCATCGAGGTCAACCCGTTCGTC ACCAAGCACACGTCGCTGCGCGGCTTCGTCTTCGATGTCGCCACCGGCAAACTCAACGAGGTCACGCCCT AG code
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * CDS Gene Sequence (paste as text only):**
 * GC% Content for gene:** 55%
 * 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)


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