EcMetAP2

**Target : Methionyl Aminopeptidase 2 (MetAP2)**
Vittaforma corneae is a human infecting microsporidia. It causes eye infections in immunocompetent people and infections in immunocompromised AIDS patients. Microsporidia can infect almost all species on earth. This specific organism can cause keratitis and systemic infections. Infection is determined through testing urine and other bodily fluids for the spores. Most current treatments do not affect the organism significantly so there is a great need for new drugs and treatments. Failure of adequate treatment results in blindness in afflicted individuals. The MetAP2 enzyme playes a role in tissue repair and possibly cell growth.
 * NCBI Gene # or RefSeq#:**
 * Protein ID (NP or XP #) or Wolbachia#:**
 * Organism (including strain):** //Vittaforma corneae//
 * Etiologic Risk Group (see link below):**
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**

MetAp2 is a part of the methionyl aminopeptidase family associated with protein coding. When the protein is inhibited, a common reaction is that the cell eventually dies. When the protein is over expressed, the result can either be cell death or over growth of cells resulting in various forms of cancer. http://www.genecards.org/cgi-bin/carddisp.pl?gene=METAP2
 * Essentiality of this protein:**


 * Complex of proteins?:** Methionyl Aminopeptidase Family

peptide + H2O = N-terminal amino acid + peptide, preferentially methionine, further reaction: arylamide + H2O = aromatic hydrocarbon + amine
 * Druggable Target:**


 * EC#: ** 3.4.11.18

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.11.18
 * Link to BRENDA EC# page:**




 * Enzyme Assay information (spectro**
 * photometric, coupled assay ?, reagents):**

The enzymatic reaction catalyzed by MetAP can be continuously

monitored in real time on a UV-VIS spectrophotometer

by simply adding excess DTNB into a MetAP reaction

mixture. MetAPs of both bacterial and eukaryotic origins

exhibit strong sequence specificity at the N-terminus,

with methionine being the most preferred amino

acid at the P1 position (3, 5, 9, 10, 24–26). At the P19

position, MetAP prefers amino acids with small side

chains (e.g., Gly, Ala, Ser, Thr, Val, Pro, and Cys).

MetAP also requires at least a tripeptide for efficient

hydrolysis, although the identity of the side chains at

P29 position and beyond does not appears to be critical

(9, 10).

http://www.sciencedirect.com/science/article/pii/S0003269700945135 Spectrophotometric assay used a custom synthesized enzyme. http://www.sigmaaldrich.com/catalog/product/aldrich/d218200?lang=en®ion=US
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**

http://www.sciencedirect.com/science/article/pii/S0003269700945135
 * -- link (or citation) to paper that contains assay information**


 * -- List cost and quantity of substrate reagents and supplier**
 * -DTNB -- $22**

Show pairwise alignment of your BLASTP search in NCBI against the PDB Query Coverage: 95% Max % Identities: 45% % Positives: 63%
 * Structure Available (PDB or Homology model)**


 * Current Inhibitors:** Fumagillin, Ovalicin

It has been expressed inhuman, rabbit, and Encephalitozoon cunicul.
 * Expression Information (has it been expressed in bacterial cells):**

Microsporidian spores were purified by filtration through nucleopore filters, treated with 0.2% SDS for 20 min, washed with PBS and resuspended in proteinase K buffer [10 mM pH 7.5 Tris HCl, 10 mM EDTA, 150 mM NaCl, 0.4% SDS]. Purified spores were then disrupted using acid-washed 500-μm glass beads in a Mini-Bead beater (Bio-spec Products Inc., Bartlesville, OK). Proteinase K was added to the disrupted spores and the solution was incubated for 15 min at 65°C. DNA was then prepared by phenol/chloroform extraction followed by ethanol precipitation.
 * Purification Method:**

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109671/

1B59- Human MetAp2 Complexed with Ovalicin
 * Image of protein (PyMol with features delineated and shown separately):**

MSFVLLNKIEPQPIEFLEPASFNLTSDSILNDALRAAEAHRRVRSSVQQILKPGVSLLQIIETVEKATRA LLVGEKNNGIGFPCGVSLNDCAAHFTLNPGDRNIILKESDILKIDFGTHSNGRIMDSAFTVCFDPKFEQL LKASQEATKRGLEVIGIDMMVCEIGREISEVFKSFEIELDNKIMPIKPIWNLNGHSIEQYRIHGEFSIPP INNGDTSRVSEGFCAIETFASTGKGEVCDKGEASHFMINRNPPSSKIYNAKNEKVLKTIQKEFGTLPFSP RHVEFYSPDSFSSIKLLSLRKFIDPYPPLHDLPNSFVAQFEHTVYLSDTGKRILTNGNDY
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**


 * length of your protein in Amino Acids:** 340


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** 79kDa

14815
 * 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.

ATGTCCTTTGTACTTCTTAACAAAATAGAGCCTCAACCAATTGAATTTCTGGAACCAGCTTCATTTAATT TAACAAGCGATTCAATATTAAATGACGCCTTACGTGCTGCAGAGGCCCATAGGCGGGTAAGGAGCAGCGT CCAACAGATTCTAAAACCAGGAGTTTCACTACTGCAAATTATAGAAACTGTGGAAAAAGCTACACGAGCT TTACTAGTAGGAGAGAAGAACAACGGAATTGGATTTCCATGTGGTGTAAGCTTAAATGACTGTGCTGCTC ACTTTACACTCAATCCAGGAGATAGGAACATAATATTAAAAGAATCTGATATTCTAAAAATAGATTTTGG AACACATTCTAATGGCAGAATTATGGATTCTGCATTTACAGTCTGTTTTGATCCAAAGTTTGAACAGTTA CTAAAGGCCTCACAAGAAGCTACCAAAAGAGGACTAGAGGTTATAGGAATTGATATGATGGTGTGTGAAA TAGGTAGGGAAATTTCTGAAGTATTCAAATCCTTTGAAATAGAGTTAGATAATAAAATCATGCCGATAAA GCCCATATGGAATCTTAACGGTCATTCTATTGAGCAATATAGAATACATGGTGAGTTTTCAATACCACCA ATCAATAATGGAGACACATCAAGAGTTTCAGAGGGATTCTGTGCCATTGAAACATTTGCAAGTACAGGAA AGGGTGAGGTCTGTGATAAAGGTGAAGCATCCCATTTTATGATCAATAGAAACCCACCAAGTTCAAAAAT TTATAATGCTAAAAATGAAAAAGTTCTTAAGACTATTCAAAAAGAGTTTGGAACATTGCCCTTTAGTCCT AGACATGTTGAGTTTTATAGCCCTGATTCATTTTCAAGTATCAAGCTGCTATCACTTAGAAAATTTATCG ACCCATACCCACCTCTTCATGATTTACCAAACTCCTTTGTTGCACAATTTGAGCATACAGTTTATCTTTC AGACACAGGGAAGAGAATACTTACAAACGGCAACGATTATTAA
 * CDS Gene Sequence (paste as text only):**


 * GC% Content for gene:**
 * 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):**

Resources:
http://www.ncbi.nlm.nih.gov/protein/429961911?report=genpept http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.11.1 http://www.ncbi.nlm.nih.gov/pubmed/12425527 http://aac.asm.org/content/52/2/790.full http://pubmedcentralcanada.ca/pmcc/articles/PMC2759695/

See **ProtocolTargetDiscoveryVDS.docx** for more Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334

@http://www.niaid.nih.gov/Pages/default.aspx @http://eupathdb.org/eupathdb/ @https://patricbrc.vbi.vt.edu/portal/portal/patric/Home @http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes @http://tubic.tju.edu.cn/deg/ @http://csgid.org/csgid/cake/pages/community_request_gateway @http://tdrtargets.org/ @http://gsc.jcvi.org/status.shtml
 * Databases of genes/organisms:**

@http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
 * Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)**

NCBI GENE Page: @http://www.ncbi.nlm.nih.gov/gene BLAST Page: @http://blast.ncbi.nlm.nih.gov/
 * Gene Information:**

NCBI Protein Page: @http://www.ncbi.nlm.nih.gov/protein Protein Expression Website Protein Expression Paper:
 * Protein Information:**


 * Primer Overlap PCR Articles**


 * Is my target good for Virtual Screening programs?**