Target+-+acetoacetyl-CoA+reductase+- Burkholderia+pseudomallei


 * *Target (protein/gene name): **acetoacetyl-CoA reductase


 * *NCBI Gene # or RefSeq#: **3693808


 * *Protein ID (NP or XP #) or Wolbachia#: **NC_007435.1


 * *Organism (including strain): **Burkholderia pseudomallei 1710b


 * Etiologic Risk Group (see link below): **CDC Category B biological threat agent


 * * Disease Information (sort of like the Intro to your Mini research write up): ** B. pseudomallei is a pathogenic gram-negative bacteria that causes a disease called melioidosis and is a soil bacteria endemic to Southeast Asia and Northern Australia. Melioidosis exists in both chronic and acute forms and it causes pain in chest, bones, or joints; cough; skin infections, lung nodules and pneumonia. This disease is really tricky to get rid of, current treatment is very expensive and long lasting and consists of an intravenous high intensity phase and an eradication phase. In the intravenous phase antibiotics are administered intravenously for a minimum of 2 weeks while the body temperature of the victim goes down to normal for 48 hours, this fever can persist for weeks or months even with appropriate treatment. In the eradication phase the patient is to take antibiotics for 12 to 20 weeks to prevent recurrence. Melioidosis comes mostly from contact with contaminated soils such as working on a rice field. Spreading melioidosis from person to person is possible but unlikely, though there are concerns that it can be engineered to be used as a biological warfare agent. If untreated melioidosis has a mortality rate that exceeds 90% and if treated with antibiotics the mortality rate is still only as low as 10% and recurrence occurs in 10-20% of patients.


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


 * Link to Gene Database page: **http://www.ncbi.nlm.nih.gov/gene/?term=Burps1710b_a1014


 * Essentiality of this protein: ** Acetoacetyl-CoA reductase is essential to pathways in butanoate metabolism which is found in many bacteria such as tuberculosis. It is responsible for the reduction of acetoacetyl-CoA into 3-oxoacyl-CoA which is a coenzyme involved in the synthesis of fatty acids.


 * Complex of proteins?: ** Acetoacetyl-CoA reductase is a t etramer in solution.

This target has been said to be a good target to search for drugs for because it has no close human homolog, is part of an essential metabolic pathway, and it has a deep binding pocket.
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **
 * http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0053851


 * *EC#: **1.1.1.36

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.36&organism= Acetoacetyl-CoA reductase + NADPH; spectrophotometric
 * Link to BRENDA EC# page: **
 * Enzyme Assay information: **
 * http://aem.asm.org/content/75/19/6168.full


 * NADPH: **
 * http://www.sigmaaldrich.com/catalog/search?interface=All&term=n6505&N=0&mode=match%20partialmax&focus=product&lang=enion=US
 * Sigma-Aldrich N6505
 * 25 mg for $90.30 or 100 mg for $275.00

PDB: 3GK3
 * Structure: **
 * http://www.rcsb.org/pdb/explore/explore.do?structureId=3GK3

SUBSTRATES INFO:
See Wu and Knight paper r for ino on NADH and NADPH degradation: Conclusions: store NADPH above pH 7.5. Can use Tris + NaOH to do this.

@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 __ __<span style="font-family: Arial,sans-serif; font-size: 13.3333px;">(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.

Enzo Life Sciences: NADPH is unstable in acid media. Stock solutions should be at pH 8. Soluble in water (50mg/ml) or 0.01M sodium hydroxide.

http://www.brenda-enzymes.info/literature/lit.php4?e=1.1.1.36&r=286615
 * Current Inhibitors: **
 * Competitively inhibited by acetyl-CoA, butyryl-CoA, methylmalonyl-CoA, and 2-methylcrotonyl-CoA
 * Sulfhydryl reagents have limited inhibition

Expressed in E. coli cells. http://oca.weizmann.ac.il/oca-bin/ocashort?id=3GK3
 * Expression Information (has it been expressed in bacterial cells): **
 * This link contains tons of information on how 3GK3 was expressed.

Can have a His-Tag and purified using Nickel column chromatography.
 * Purification Method : **

>3GK3:A|PDBID|CHAIN|SEQUENCE MAHHHHHHMGTLEAQTQGPGSMQAKRVAFVTGGMGGLGAAISRRLHDAGMAVAVSHSERNDHVSTWLMHERDAGRDFKAY AVDVADFESCERCAEKVLADFGKVDVLINNAGITRDATFMKMTKGDWDAVMRTDLDAMFNVTKQFIAGMVERRFGRIVNI GSVNGSRGAFGQANYASAKAGIHGFTKTLALETAKRGITVNTVSPGYLATAMVEAVPQDVLEAKILPQIPVGRLGRPDEV AALIAFLCSDDAGFVTGADLAINGGMHMS >3GK3:B|PDBID|CHAIN|SEQUENCE MAHHHHHHMGTLEAQTQGPGSMQAKRVAFVTGGMGGLGAAISRRLHDAGMAVAVSHSERNDHVSTWLMHERDAGRDFKAY AVDVADFESCERCAEKVLADFGKVDVLINNAGITRDATFMKMTKGDWDAVMRTDLDAMFNVTKQFIAGMVERRFGRIVNI GSVNGSRGAFGQANYASAKAGIHGFTKTLALETAKRGITVNTVSPGYLATAMVEAVPQDVLEAKILPQIPVGRLGRPDEV AALIAFLCSDDAGFVTGADLAINGGMHMS >3GK3:C|PDBID|CHAIN|SEQUENCE MAHHHHHHMGTLEAQTQGPGSMQAKRVAFVTGGMGGLGAAISRRLHDAGMAVAVSHSERNDHVSTWLMHERDAGRDFKAY AVDVADFESCERCAEKVLADFGKVDVLINNAGITRDATFMKMTKGDWDAVMRTDLDAMFNVTKQFIAGMVERRFGRIVNI GSVNGSRGAFGQANYASAKAGIHGFTKTLALETAKRGITVNTVSPGYLATAMVEAVPQDVLEAKILPQIPVGRLGRPDEV AALIAFLCSDDAGFVTGADLAINGGMHMS >3GK3:D|PDBID|CHAIN|SEQUENCE MAHHHHHHMGTLEAQTQGPGSMQAKRVAFVTGGMGGLGAAISRRLHDAGMAVAVSHSERNDHVSTWLMHERDAGRDFKAY AVDVADFESCERCAEKVLADFGKVDVLINNAGITRDATFMKMTKGDWDAVMRTDLDAMFNVTKQFIAGMVERRFGRIVNI GSVNGSRGAFGQANYASAKAGIHGFTKTLALETAKRGITVNTVSPGYLATAMVEAVPQDVLEAKILPQIPVGRLGRPDEV AALIAFLCSDDAGFVTGADLAINGGMHMS
 * Image of protein: **
 * *Amino Acid Sequence: **


 * *length of your protein in Amino Acids: **1076 Amino Acids


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: **114909.0 kD


 * Molar Extinction coefficient of your protein at 280 nm wavelength: **62630 M-1 cm-1


 * TMpred graph Image: **

>fid|19242473|locus|VBIBurPse115837_5074| Acetoacetyl-CoA reductase (EC 1.1.1.36) [Burkholderia pseudomallei 1710b] atgcaagcaaaacgcgtggcattcgtgacgggcggtatgggcggactgggtgcggcgatc agccggcggctgcacgacgccggcatggctgtggcggtgtcgcattcggagcgcaacgat catgtgtcgacgtggctgatgcacgagcgcgacgccggacgcgatttcaaggcgtatgcg gtcgacgtcgcggatttcgaatcgtgcgagcggtgcgcggagaaggtgctcgccgatttc ggcaaggtcgacgtgctgatcaacaacgcggggatcacgcgcgatgcgacgttcatgaag atgacgaagggcgactgggacgcggtgatgcgcaccgatctcgatgcgatgttcaacgtg acgaaacagttcatcgcgggcatggtcgagcggcgcttcgggcgcatcgtcaacatcggt tcggtgaacggctcgcgcggcgcgttcggtcaggcgaattatgcgtcggcgaaggcgggc atccacggtttcacgaagacgctcgcgctcgagacggcgaagcgcgggatcacggtcaac acggtgtcgcccggctatctcgcgaccgcgatggtcgaagcggtgccgcaggacgtgctc gaggcgaagatcctgccgcagattccggttggccggctcggccggcccgacgaagtggcc gcgctcatcgcgtttctgtgctcggacgacgcggggttcgtcaccggcgccgatctcgcg atcaacggcgggatgcacatgtcctga
 * *CDS Gene Sequence (paste as text only): **


 * *GC% Content for gene: ** 65.19% as determined by http://www.endmemo.com/bio/gc.php GC content calculator.

ATGCAGGCCA AGCGTGTGGC GTTCGTAACC GGCGGTATGG GCGGCTTAGG CGCCGCGATT TCCCGTCGTC TTCATGATGC CGGAATGGCG GTCGCCGTTA GTCATTCTGA GCGCAACGAT CATGTGTCTA CCTGGCTCAT GCACGAACGT GACGCGGGCC GTGACTTTAA AGCGTATGCA GTTGACGTGG CCGATTTTGA GAGCTGCGAA CGGTGTGCCG AGAAAGTGCT GGCGGACTTT GGCAAAGTAG ACGTGTTGAT TAACAACGCG GGAATTACTC GTGATGCCAC CTTCATGAAA ATGACAAAGG GTGATTGGGA CGCTGTTATG CGTACAGATC TGGACGCCAT GTTTAACGTA ACCAAACAGT TTATTGCCGG TATGGTGGAA CGTCGCTTCG GTCGTATCGT TAACATTGGT AGCGTTAACG GGTCGCGCGG CGCGTTTGGA CAAGCCAACT ACGCCAGTGC AAAAGCCGGC ATCCATGGCT TTACGAAGAC GTTAGCCCTG GAGACAGCAA AACGTGGGAT CACCGTCAAT ACCGTCAGCC CGGGTTATTT GGCGACGGCT ATGGTTGAAG CAGTCCCTCA AGATGTTCTC GAAGCAAAAA TTCTGCCACA GATTCCCGTT GGTCGTCTGG GTCGTCCAGA TGAAGTGGCC GCGCTGATTG CGTTCCTGTG TTCGGATGAC GCCGGTTTCG TGACGGGGGC TGACCTCGCA ATCAACGGGG GGATGCATAT GTCATGA
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **

This was determined using Integrated DNA Technologies, Inc. Codon Optimizer with E. coli as the selected organism. The first result was recorded.
 * https://www.idtdna.com/CodonOpt


 * *GC% Content for gene (codon optimized): ** 54.48% as determined by http://www.endmemo.com/bio/gc.php GC content calculator.


 * Primer design results for 'tail' primers: **