TargetSp15+-+Disulfide+Oxidoreductase+(Burkholderia+Pseudomallei)

**NCBI Gene # or RefSeq#:** 3093150 **Protein ID (NP or XP #) or Wolbachia#:** NC_006350.1 **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):** Melioidosis, also called Whitmore's disease, is an infectious disease that can infect humans or animals. The disease is caused by the bacterium //Burkholderia pseudomallei //. 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 can cause both chronic and acute forms and it causes pain in chest, bones, or joints; cough; skin infections, lung nodules and pneumonia. The current treatment of the disease is expensive and generally starts with intravenous (within a vein) antimicrobial therapy for 10-14 days followed by a long dosage of oral antibiotics . Melioidosis comes mostly from contact with contaminated soils such as working on a rice field. Melioidosis can also 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 as low as 10% and recurrence occurs in 10-20% of patients. **Link to TDR Targets page (if present):** n/a **Link to Gene Database page:** **Essentiality of this protein:** DSB proteins play a role in bacterial and are essential for the proper folding and activity of a range of virulence factors in a number of bacterial species including clinically significant pathogens such as //Pseudomonas aeruginosa//, enteropathogenic //Escherichia coli//, and //Neisseria meningitidis//. DsbA presents an opportunity to disrupt multiple downstream virulence effectors //via// its inhibition. Interrogation of the sequenced genome of //B. pseudomallei// identified a DsbA homologue, prompting investigation for it as a potential essential mediator of virulence in this pathogen. This study was undertaken in the paper “Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo. ” []
 * Target (protein/gene name): **Disulfide Oxidoreductase (DsbA)
 * Organism (including strain): ** Burkholderia Pseudomallei (strain K96243)


 * Is it a m **** onomer or multimer as biological unit **** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): structure is monometric.

**Complex of proteins?:** <span style="font-family: Verdana,sans-serif; font-size: 13.3333330154419px; line-height: 20px;">No <span style="font-family: Verdana,sans-serif; font-size: 10pt;"> **Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** Data presented in paper “Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo.” Suggested that DsbA is an essential mediator of virulence in //B. pseudomallei//and that its disruption has pleiotropic effects, validating it as a promising drug target in the organism tested (mice). <span style="font-family: Verdana,sans-serif; font-size: 10pt; line-height: 1.5;">[]


 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">*EC#: **<span style="font-family: Verdana,sans-serif; font-size: 10pt;">1.8.4.2 potential EC number multiple were given however none of the ECs listed related to the organism of interest B<span style="font-family: Verdana,sans-serif; font-size: 13.3333330154419px;">urkholderia Pseudomalle.

<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> **Link to BRENDA EC# page:** <span style="font-family: Verdana,sans-serif; font-size: 10pt;">[]



2 glutathione + protein-disulfide = glutathione-disulfide + protein-dithiol

<span style="font-family: Verdana,sans-serif; font-size: 10pt;">Determined an applicable EC number as although the disuphide oxidoreductase is shown it does not correspond to the organism Burkholderia pseudomallei but instead corresponds to the organism Pseudomonas aeruginosa. This is allowed as when run through the program Blast Pro Pseudomonas aeruginosa came up as a similar organism and it was also mentioned in the paper “Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo.” about their alikeness.

<span style="font-family: Verdana,sans-serif; font-size: 10pt;">For crystallization experiments and all biochemical characterizations (except the insulin reduction assay) the N-terminal His 6-tagged BpsDsbA construct was expressed and purified as previously described for PaDsbA. For the insulin reduction assay, the C-terminal His6-tag construct **was expressed in****<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> //E. coli// BL21 (DE3) **<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> at 310 K under isopropyl β-D-thiogalactopyranoside induction and purified using immobilized metal affinity chromatography. Additional details of cloning, expression, and purification methods are provided in Supplementary Data. <span style="font-family: Arial,sans-serif; font-size: 10pt;">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901323/
 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">Purification Method and expression: **<span style="font-family: Verdana,sans-serif; font-size: 10pt;">Can have a His-Tag and purified using Nickel column chromatography.


 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">PDB protein file: **<span style="font-family: Verdana,sans-serif; font-size: 10pt;">4k2d http://www.rcsb.org/pdb/explore/explore.do?structureId=4k2d

<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> **Image of protein:**


 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">*Amino Acid Sequence: **
 * **<span style="font-family: 'Times New Roman',serif; font-size: 9pt; line-height: 1.5;">1 ** ||  || <span style="font-family: 'Times New Roman',serif; font-size: 9pt;">SNAAGFAQAS PSAPVAGKDF EVMKSPQPVS APAGKVEVIE FFWYGCPHCY ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 9pt;">51 ** ||  || <span style="font-family: 'Times New Roman',serif; font-size: 9pt;">EFEPTIEAWV KKQGDKIAFK RVPVAFRDDF VPHSKLFYAL AALGVSEKVT ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 9pt;">101 ** ||  || <span style="font-family: 'Times New Roman',serif; font-size: 9pt;">PAVFNAIHKE KNYLLTPQAQ ADFLATQGVD KKKFLDAYNS FSVQGQVKQS ||
 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">*length of your protein in Amino Acids: **<span style="font-family: Verdana,sans-serif; font-size: 10pt;">200 Amino Acids

<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> **Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** <span style="font-family: Verdana,sans-serif; font-size: 11pt;">21996.1 <span style="font-family: Verdana,sans-serif; font-size: 10pt;"> kD

<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> **Molar Extinction coefficient of your protein at 280 nm wavelength:** <span style="font-family: Verdana,sans-serif; font-size: 11pt;">23045 <span style="font-family: Verdana,sans-serif; font-size: 10pt;">M-1 cm-1


 * <span style="font-family: Verdana,sans-serif; font-size: 10pt;">TMpred graph Image **<span style="font-family: Verdana,sans-serif; font-size: 10pt;"> (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * <span style="font-family: Verdana,sans-serif;">Figure 3 **<span style="font-family: Verdana,sans-serif;"> two possible models considered, only significant TM-segments used - probably no transmembrane protein - no possible model found

http://www.bioinformatics.org/sms2/rev_trans.html <span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium; line-height: 1.5;">>reverse translation of Untitled to a 450 base sequence of most likely codons. agcaacgcggcgggctttgcgcaggcgagcccgagcgcgccggtggcgggcaaagatttt gaagtgatgaaaagcccgcagccggtgagcgcgccggcgggcaaagtggaagtgattgaa tttttttggtatggctgcccgcattgctatgaatttgaaccgaccattgaagcgtgggtg aaaaaacagggcgataaaattgcgtttaaacgcgtgccggtggcgtttcgcgatgatttt gtgccgcatagcaaactgttttatgcgctggcggcgctgggcgtgagcgaaaaagtgacc ccggcggtgtttaacgcgattcataaagaaaaaaactatctgctgaccccgcaggcgcag gcggattttctggcgacccagggcgtggataaaaaaaaatttctggatgcgtataacagc <span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium; line-height: 1.5;">tttagcgtgcagggccaggtgaaacagagc
 * *CDS Gene Sequence (paste as text only): - using **


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Assay Data: **

<span style="font-family: Verdana,sans-serif; font-size: 10pt;">Insulin reduction assay:

<span style="font-family: Verdana,sans-serif; font-size: 10pt;">The insulin reduction assay was carried out as previously described ( [|35] ). Briefly, reaction mixtures were prepared containing 8 μM recombinant BpsDsbA or recombinant thioredoxin from E. coli 0.1 M phosphate buffer, 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.35 mM DTT. The reactions were started by adding insulin to a final concentration of 131 μM and the reduction of insulin was monitored at A650 for 80 min at 20 s intervals using a Shimadzu 1800 UV/visible spectrophotometer. The non-catalyzed reduction of insulin by DTT was monitored in a control reaction without catalyst.


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901323/ **


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Pricing: **

Glutathione 500mg $52.60

[]