Target+-+Fab+G+3-ketoacyl-(acyl-carrier-protein)+reductase

** *Target (protein/gene name): ** Fab G 3-ketoacyl-(acyl-carrier-protein) reductase

 * NCBI Gene # or RefSeq#:** 883564


 * Protein ID (NP or XP #) or Wolbachia#:** NP_221114.1


 * Organism (including strain):** Rickettsia prowazekii


 * Etiologic Risk Group (see link below):** Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia

Epidemic typhus is caused by Rickettsia prowazekii bacteria and is spread to humans through lice or ticks. In the US, the bacterium is most commonly found on the flying squirrel. Contraction of typhus can be prevented through a series of vaccines. It is uncommon in the US, but a few cases of epidemic typhus have been reported in Austin, Texas. Typhus is more common in areas of the world that have low hygiene and cold temperatures. Once the disease has been contracted, it can become dormant and then re-activate. This is known as Brill-Zinsser disease and is more common in the elderly. If left untreated, the disease has the potential to be fatal. Death occurs in 10-60% of patients. Symptoms include chills, confusion, stupor, rash, high fever, cough, headache, arthralgia, low blood pressure, severe headache and muscle pain. A CBC (complete blood count) can be used to diagnose the disease and shows levels of typhus antibodies, levels albumin, sodium level, and liver enzyme levels. Common treatments include antibiotics such as tetracycline. Prevention measures include bathing, avoiding areas that are infested with rat fleas or lice, boiling infected clothing for a period of 5 days, and using insecticides.
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**


 * Link to TDR Targets page (if present):** Not present


 * Link to Gene Database page:** http://www.ncbi.nlm.nih.gov/gene/883564


 * Essentiality of this protein:** Very essential in the biosynthesis of fatty acids pathway
 * Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25A° resolution
 * http://www.ssgcid.org/static/media/uploads/publications/PUBL_ActaF_24_en5463_Subramanian_kaACP.pdf


 * Complex of proteins?:** Tetramer in solution


 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):**
 * Inhibition of Plasmodium falciparum Fatty Acid Biosynthesis: Evaluation of FabG, FabZ, and FabI as Drug Targets for Flavonoids
 * Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25A° resolution
 * http://www.ssgcid.org/static/media/uploads/publications/PUBL_ActaF_24_en5463_Subramanian_kaACP.pdf


 * *EC#: ** 1.1.1.100

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.100
 * Link to BRENDA EC# page:**



(3R)-3-hrdroxyacyl-[acyl-carrier protein] plus NADP+; spectrophotometric
 * Enzyme Assay information:**


 * Substrate Info (Dr. B') - 100113**
 * May also dilute in Tris - talk to Kaarthik.**

@http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf b -NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution. It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 ° C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are b -NADP and monophosphoadenosine 5 ¢ - diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, __it has been__ __reported that a 0.5 mM solution in 0.02 M NaOH __ __(pH 12.3) showed no loss of purity in a week at 4 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C or __ __ - <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">85 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C, but a 13% loss at –20 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C. <span style="font-family: Arial,sans-serif; font-size: 9.33333px;">3 __
 * NADPH** - sigma N6505
 * <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">Preparation Instructions **
 * <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">Storage/Stability **

For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.

PDB ID: 3F9I
 * Structure Available (PDB or Homology model)**


 * Current Inhibitors:** Flavonoids, EGCG in green tea


 * Expression Information (has it been expressed in bacterial cells):** Expressed in E. coli cells


 * Purification Method:** Nickel column + FPLC


 * Image of protein:**



MIDLTGKTSLITGASSGIGSAIARLLHKLGSKVIISGSNEEKLKSLGNALKDNYTIEVCN LANKEECSNLISKTSNLDILVCNAGITSDTLAIRMKDQDFDKVIDINLKANFILNREAIK KMIQKRYGRIINISSIVGIAGNPGQANYCASKAGLIGMTKSLSYEVATRGITVNAVAPGF IKSDMTDKLNEKQREAIVQKIPLGTYGIPEDVAYAVAFLASNNASYITGQTLHVNGGMLM VX
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**


 * Length of your protein in Amino Acids:** 242


 * Molecular Weight in kiloDaltons:** 25870.1 kD


 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 10680 M-1 cm-1, 10430 M-1 cm-1


 * TMpred graph Image **:



CAAACAATATTTAAAAGAACATCCACAAATTTCCAACGAAATTGAGAAAATAATACGAGAAAAATCGTCA GCAATTACTAATATAAATCTTGATCAAACGGAGGAATAAAATGATTGATTTAACAGGTAAAACTTCCTTA ATTACAGGTGCTTCAAGTGGCATAGGAAGTGCCATAGCGCGGTTACTACATAAACTCGGGAGCAAGGTAA TTATTAGCGGTAGTAATGAAGAAAAATTAAAATCACTTGGCAATGCTTTAAAAGATAATTATACCATAGA AGTATGTAATCTTGCGAATAAGGAAGAATGTAGTAATTTAATATCTAAAACATCAAATTTAGATATTTTA GTATGTAATGCCGGTATTACTAGTGATACACTCGCAATTAGAATGAAAGATCAAGATTTCGACAAAGTTA TTGATATTAATTTGAAAGCCAATTTTATTTTAAATCGTGAAGCAATAAAAAAAATGATACAAAAAAGGTA TGGACGCATTATTAACATATCTTCAATAGTAGGAATTGCAGGTAACCCAGGACAGGCTAATTATTGCGCT TCTAAAGCAGGTTTAATAGGTATGACCAAATCGCTATCTTACGAAGTTGCAACTAGAGGGATTACGGTTA ATGCAGTAGCACCAGGATTTATTAAATCAGATATGACTGATAAACTCAATGAAAAACAACGAGAAGCAAT AGTACAAAAAATTCCACTAGGTACTTATGGTATACCAGAAGACGTTGCATACGCAGTCGCATTTTTAGCA AGCAATAATGCATCATATATTACTGGTCAAACTCTACATGTAAACGGTGGTATGTTAATGGTATAAAATT TTATGCTAGCAAAAAATAATTTTTTGTGCTAGAAGTTCTTTTAAAGAGAAAATGCTTATGAATATTTATT TCAAATATTCATAGCAATGAAAATAAAATCTAAT
 * CDS Gene Sequence (paste as text only):**


 * GC% Content for gene:** 30.6%

ATGATTGACCTCACGGGCAAAACTTCTCTGATCACGGGTGCCAGCTCTGGTATCGGTTCT GCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTATCTCTGGTTCTAACGAA GAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGTTTGTAAC CTCGCGAACAAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTG GTTTGCAATGCGGGCATCACCTCTGACACCCTGGCGATCCGTATGAAAGACCAGGACTTT GACAAAGTCATCGATATCAACCTGAAAGCGAACTTCATCCTGAACCGTGAAGCGATCAAG AAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCTATCGTTGGTATTGCG GGTAACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAA TCTCTGTCTTACGAAGTTGCGACCCGCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTC ATCAAATCTGACATGACTGACAAGCTGAATGAAAAACAGCGTGAGGCAATCGTTCAGAAA ATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTACGCGGTTGCGTTCCTGGCG TCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGATG GTTTAA
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized):** 48.6%

1 ATGATTGACCTCACGGGCAAA 21 2 GCAGAACCGATACCAGAGCTGGCACCCGTGATCAGAGAAGTTTTGCCCGTGAGGTCAATC 60 3 GCTCTGGTATCGGTTCTGCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTA 60 4 GAGAGACTTCAGTTTTTCTTCGTTAGAACCAGAGATAATAACTTTAGAGCCCAGTTTGTG 60 5 TAACGAAGAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGT 60 6 ATCAGATTGCTGCATTCTTCTTTGTTCGCGAGGTTACAAACTTCGATGGTGTAGTTGTCT 60 7 AAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTGGTTTGCAAT 60 8 CTTTCATACGGATCGCCAGGGTGTCAGAGGTGATGCCCGCATTGCAAACCAGGATGTCCA 60 9 CCTGGCGATCCGTATGAAAGACCAGGACTTTGACAAAGTCATCGATATCAACCTGAAAGC 60 10 CATCTTCTTGATCGCTTCACGGTTCAGGATGAAGTTCGCTTTCAGGTTGATATCGATGAC 60 11 CGTGAAGCGATCAAGAAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCT 60 12 GTTCGCCTGACCTGGGTTACCCGCAATACCAACGATAGAGGAGATGTTGATAATACGACC 60 13 ACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAATCTC 60 14 CGTTGACGGTGATACCGCGGGTCGCAACTTCGTAAGACAGAGATTTGGTCATGCCGATCA 60 15 GCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTCATCAAATCTGACATGACTGACAAGC 60 16 GATTTTCTGAACGATTGCCTCACGCTGTTTTTCATTCAGCTTGTCAGTCATGTCAGATTT 60 17 TGAGGCAATCGTTCAGAAAATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTA 60 18 TGTAAGACGCGTTGTTAGACGCCAGGAACGCAACCGCGTAAGCCACGTCTTCTGGAATAC 60 19 CGTCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGA 60 20 TTAAACCATCAGCATACCACCGTTAACAT 29
 * Primer design results for pNIC-Bsa4 cloning:**

https://docs.google.com/file/d/0B_Gl3lMyhDsoQ0pkekVLcHZTVVE/edit?usp=sharing
 * DNAWorks Output Link:**

<span style="color: #000000; font-family: Arial,Helvetica,sans-serif;">Forward Primer: <span style="color: #2e9fae; font-family: Arial,Helvetica,sans-serif;">5’ TACTTCCAATCCATGATTGACCTCACGGGCAA 3'
 * Primer design results for 'tail' primers (this is just 2 sequences):**

<span style="color: #000000; font-family: Arial,Helvetica,sans-serif;">Reverse Primer:

<span style="color: #2e9fae; font-family: Arial,Helvetica,sans-serif;">5’ GTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’