TargetSp15-+Enoyl-ACP+reductase+(Rickettsia+rickettsia)

Target (protein/gene name): acetylglutamate kinase *NCBI Gene # or RefSeq#: 5850264 *Protein ID (NP or XP #) or Wolbachia#: WP_012150360 *Organism (including strain):Rickettsia rickettsii (str. Iowa) Etiologic Risk Group (see link below): Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia */Disease Information (sort of like the Intro to your Mini Research Write up): R. rickettsia is a unicellular, Gram-negative coccobacillus that is native to the Americas. It is known to cause Rocky Mountain Spotted Fever (RMSF), a disease that can be lethal if not treated. RMSF symptoms include fever, headache, pain, and a rash. R. rickettsii is spread by Dermacentor Ticks. Even when treated with current antibiotics, some patients can require hospitalization because R. rickettsii infects the lining of the blood vessels, which may damage the respiratory system, the central nervous system, gastrointestinal system, or kidneys. If the infection is severe enough there is a chance for partial paralysis of the lower extremities, gangrene (may require amputation), and loss of hearing and bladder control. Research of //R. rickettsii// started when early settles of Western Montana began to be plagued with a disease that they called “black measles” because of its severe dark rash. Fatalities occurred in nearly 4 out of 5 adult cases. This caused the Montana State Board of Health to bring in scientists to investigate the cause and treatment for the spotted fever. One such scientist was Dr. Howard Ricketts, a pathologist, who in 1906 had discovered that the vector for the disease was the Rocky Mountain wood tick. Then in 1909 he had isolated the bacterium that was responsible for the disease, which was later named //Rickettsia rickettsii// in his honor. This spawned the building of The Rocky Mountain Labs, one of the few Biosafety level 4 laboratories. []

Link to TDR Targets page (if present): []

Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): []

Essentiality of this protein: Acetlyglutamate kinase catalyzes the second and frequently controlling step of arginine synthesis which is key for meaning cellular functions.

[] Is it a monomer or multimer as biological unit? (make prediction at __ http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html __ ): monomer

Complex of proteins?: No

Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Druggability index: 0.2 [] *EC#: ** 2.7.2.8 ** Link to BRENDA EC# page: [] -- Show screenshot of BRENDA enzyme mechanism schematic

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 # Link to info for assay information: [] Reagents: ATP and N-acetyl-L-glutamate ATP assay kit for 100 assays: $375 Couldn’t find the rest

Structure (PDB or Homology model): Homolog ID: 1OH9

-- PDB # or closest PDB entry if using homology model: 1OH9

-- For Homology Model option:

Show pairwise alignment of your BLASTP search in NCBI against the PDB Query 211 DINLLKSMLDDSNNFIEEALIKIAVNALENNNGYVHFVNSEAPNSILSTM 260 D+N K + D N F +L+ IA+ L+ NG V FV+S+A N S+ Sbjct 99 DVNAWKKLYD-INFFSIVSLVGIALPELKKTNGNVVFVSSDACNMYFSSW 147 Query Coverage: 12%

Max % Identities: 39%

% Positives 55%

Chain used for homology: A Chain Amino Acid Sequence for Homolog: MGKVILVTGVSRGIGKSIVDVLFSLDKDTVVYGVARSEAPLKKLKEKYGDRFFYVVGDITEDSVLKQLVN AAVKGHGKIDSLVANAGVLEPVQNVNEIDVNAWKKLYDINFFSIVSLVGIALPELKKTNGNVVFVSSDAC NMYFSSWGAYGSSKAALNHFAMTLANEERQVKAIAVAPGIVDTDMQVNIRENVGPSSMSAEQLKMFRGLK ENNQLLDSSVPATVYAKLALHGIPDGVNGQYLSYNDPALADFMP

Current Inhibitors: None were found in www.bindingdb.com

Expression Information (has it been expressed in bacterial cells): Yes it has been expressed inEscherichia coli k12- [] Purification Method: View above link. Image of protein (PyMol with features delineated and shown separately):

*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKNQAIVLKLPATIIIDDKLFTAFIESVRLLEMCGAKIYIVHDHIDLRSSSLVSQIDENFSQKISKISDY SSLNNPIIMEILSSYVNKLIVTKLSSIGCYAVGISGKDANLLQAKKSKLSYRKIVNHDVINIAFLSEPIL INPEILLNFADNNIIPVIAPFASDDQEKTHLLNVNLTLATIASALSAEHLILPYEILQVSETFPYNIKIR DINLLKSMLDDSNNFIEEALIKIAVNALENNNGYVHFVNSEAPNSILSTMFDININ

*length of your protein in Amino Acids: 285 residues Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: 29631.3

Molar Extinction coefficient of your protein at 280 nm wavelength: Not reduced-12045 Reduced-11920

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): TTGAAGAATCAAGCAATAGTTCTAAAATTACCTGCTACTATTATTATTGACGATAAATTATTTACGGCTT TTATTGAATCTGTTAGATTGCTTGAGATGTGCGGTGCTAAAATATATATAGTACATGATCATATTGATTTACGAAGTTCGTCTTTAGTATCACAAATAGATGAAAATTTTAGTCAAAAAATTAGTAAAATAAGCGACTATAGTTCTCTAAATAATCCTATTATTATGGAAATTTTATCCAGTTACGTTAACAAGCTTATAGTAACAAAGTTAAGTAGTATAGGTTGTTATGCAGTTGGTATTTCAGGTAAGGATGCTAATTTACTGCAAGCAAAAAAATCAAAACTATCTTACCGAAAAATTGTAAATCATGATGTTATAAATATTGCTTTTTTAAGCGAGCCTATTCTGATTAATCCAGAAATTTTATTAAATTTTGCAGATAATAATATTATACCCGTGATAGCACCGTTTGCGAGCGACGATCAGGAGAAAACGCATTTATTAAATGTTAACTTAACGTTAGCAACAATTGCTTCTGCATTAAGTGCAGAACATTTAATATTGCCATATGAGATATTACAGGTATCCGAGACGTTTCCGTATAATATAAAAATACGAGATATTAACTTGTTGAAATCAATGTTAGATGATAGTAATAATTTCATCGAAGAAGCATTAATTAAGATAGCAGTTAATGCCCTTGAAAATAATAATGGTTACGTACATTTTGTGAATAGTGAAGCACCGAATTCAATATTGTCAACTATGTTTGATATTAATATAAATTAA

*GC% Content for gene: 28.5%

*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Could not find.

*GC% Content for gene (codon optimized): Could not find. 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 text file - that should be saved in your Google Docs 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): Did not do.

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