TargetSp15- 2-dehydro-3-deoxyphosphooctonate+aldolase+(Burkholderia+pseudomallei)


 * Target (protein/gene name):** 2-dehydro-3-deoxyphosphooctonate aldolase (KDOP synthase); kdsA


 * NCBI Gene # or RefSeq#:** BURPS1710b_3264


 * Protein ID #:** 3UND


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


 * Etiologic Risk Group:** NIH rating of Risk Group Level 3 for //B.pseudomallei//


 * Disease Information:** //Burkholderia pseudomallei—//a gram-negative bacterium—is the cause of a serious infectious disease known as melioidosis, or as Whitemore’s disease. Exposure to contaminated water or soil, followed by inhalation, ingestion, or contact with skin abrasions, accounts for most of the causes of transmission. Very few cases of human-to-human transfer of Melioidosis have been observed. Melioidosis is prominent in southeast Asia and in northern Australia, where it accounts for approximately 50% of sepsis-induced death. Current treatments are time and resource-laden, consisting of antimicrobial IV therapy requiring administration for 10-14 days in a hospital or clinical setting, followed by oral therapy required for 3-6 consecutive months. Yet this treatment regimen maintains high mortality rates at around 35% [1]. Treatment with more common antimicrobial agents, such as tetracyclines, macrolides, and fluoroquinolones is not effective due to B.pseudomallei’s ability to pump out such drugs.


 * Link to TDR Targets page (if present):** none available

[] [] [] []
 * Link to Gene Database page:**
 * Link to Protein Databases: **


 * Essentiality of this protein:** No essentiality data is available for 3UND in //B.pseudomallei//. However, it is reported that a homologous protein, 2-dehydro-3-deoxyphosphooctonate aldolase, found in the closely related sister species— //B. thailandensis // ( gene BTH_I1893)—is essential [2]. The protein encoded by BTH_I1893 shows query coverage of 98%, maximum identity of 51%, and positives of 67% between the amino acid sequences of //B.thailandensis// and //B.pseudomallei// (see Figure 2 below). The high degree of homology observed in the primary amino acid sequence of each species’ aldolases provides support for comparison of protein essentiality across species. Additionally, // B.thailandensis // and //B.pseudomallei// are closely related species wherein //B.thailandensis// is often considered to be a less virulent strain of //B.pseudomallei//. For this reason, it is assumed that 3UND is also essential in //B.pseudomallei//. 3UND is involved in lipopolysaccharide biosynthesis, catalyzing the following reaction:


 * __ Figure __****__ 1 __****- 3UND catalyzes the reaction between phosphoenolpyruvate and D-arabinose 5 phosphate, producing 2-dehydro-3-deoxy-D-octonate 8-phosphate and phosphate in the presence of water.**
 * __ Figure __****__ 2 __****__ - __** **A Basic Local Alignment Search Tool (BLAST) provided by NCBI was used to determine the homology between** //**B.pseudomallei and B. thailandensis **// ** . The A chain from ** ** 2-dehydro-3-deoxyphosphooctonate aldolase of each organism was used. **


 * Is it a monomer or multimer as biological unit?** 3UND is a tetrameter (multimer) as a biological unit.


 * Complex of proteins:** 3UND is a tetramer with identical A, B, C, and D subuntis of 281 amino acids per unit. However, structural analysis in PyMol indicates that even as a tetramer, A, B, C, and D possess individual active sites.
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** 3UND is cited as having no “close” human homolog, making it a prime target for //B.pseudomallei// treatment. BRENDA documents more than 18 inhibitors for 3UND, however, only (Z,E)-D-Glucophosphoenolpyruvate has been found to specifically inhibit 3UND produced by/whithin gram-negative bacteria. (Note: //Burkholderia pseudomallei // is a gram-negative bacterium).


 * EC#:** 2.5.1.55

[] **Link for //B.thailandensis//-** []
 * Link to BRENDA EC# page:**
 * Link for //B.pseudomallei//- **
 * -- ** See above for screenshot of BRENDA enzyme mechanism schematic

[]
 * Enzyme Assay information:**
 * KDO8P (Aldolase) kinetic enzyme assay-** “The standard assay was performed at 60C in a final volume of 200 ul reaction buffer consisting of 100 mM HEPES buffer pH 7.0, 0.48 mM Cd2+, 2 mM PEP, 2 mM A5P, 0.2 mM EDTA, and appropriately diluted enzyme (typically 0.3 uM). An aliquot of the enzyme (10 ul) was first preincubated with EDTA (10 ul, 4 mM) for 15 min at room temperature. To this solution was added the reaction buffer (160 ul) containing Cd2+ (0.48 mM) and the resulting mixture was incubated for 10 min at 60 C. The reaction was initiated by adding the mixture (20 ul) of both substrates, PEP (20 mM) and A5P (20 mM). After incubation at 60 C (typically for 10 min) the reaction was quenched by adding TCA to a final concentration of 5%.”
 * -- link to page paper containing assay information:**

A5P: [] PEP: []
 * -- links to assay reagents (substrates) pages.**
 * --- List cost and quantity of substrate reagents, supplier, and catalog #**

-- PDB #: 3UND [] A BLASTP of the 3UND amino acid sequence compared to the human database of amino acids yielded “no significant similarity found” between 3UND and human proteins. While this finding could be an error, the lack of similarity is supported by the claim made by Baugh, Gallagher, Patrapuvich, et. al. that KDOP synthase lacks a close human homolog[1]. This finding suggests that targeting KDOP (3UND) may be a safe drug target.
 * Quantity || Cost || Supplier || Catalog # ||
 * D-arabinose 5-phosphate disodium salt || 25 MG || $643.00 || Sigma-Aldrich ||  [|A2013] ||
 * ==Phospho(enol)pyruvic acid monopotassium salt == || 250 MG || $171.00 || Sigma-Aldrich || 860077 ||
 * Structure **
 * __ Figure __****__ 3 __****__ - __** **BLASTP homology search result between 3UND and the H.sapien protein databank. The A chain of 3UND was used (amino acid length: 281).**

According to BRENDA, the following ligands have been found as inhibitors to 3UND: (Z,E)-D-Glucophosphoenolpyruvate 1,10-phenanthroline, 1-carboxyheptane-1,7-diyl bis(phosphate) 2,6-Anhydro-3-deoxy-2beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate 2,6-pyridine dicarboxylic acid 2-dehydro-3-deoxy-D-octonate 8-phosphate 2-Deoxy-2-fluoro-D-arabinoate-5-phosphate Bromopyruvate D-arabinose 5-phosphate dipicolinic acid EDTA However, (Z,E)-D-Glucophosphoenolpyruvate is the only ligand listed above that has been observed to bind to 3UND in gram negative bacteria. Six ligands have been shown to inhibit 3UND according to BindingDB, including: CHEMBL1938424 (56950652) CHEMBL95764 (3496) [|CHEMBL1941140] (57400865) [|CHEMBL1941138] (444348) [|CHEMBL1941139] (57393902) [|CHEMBL1235229] (5326965)
 * Current Inhibitors:**


 * Expression Information:** 3UND is required for the synthesis of lipopolysaccharides, which are essential components of the outer membranes of gram-negative bacteria such as //Burkholderia pseudomallei. // KDOP synthetases are structurally conserved, and it has been anticipated that inhibitors to these enzymes will be effective across many species of //Burkholderia//, many of which are responsible for various infectious diseases, including cystic fibrosis infections and meliodosis infections [3]. Synthesis of biological membranes is a requirement of bacterial reproduction, meaning the inhibition of 3UND enzyme could prevent new membrane formation, thus preventing bacterial growth. In rapidly dividing //B.pseudomallei//—such as when bacteria are approaching log phase growth upon infecting a susceptible host—3UND would thus be expressed constitutively. The constitutive expression of BURPS1710b_3264, resulting in the synthesis of 3UND, suggests that a drug which targets 3UND may be highly effective in stopping infections. This is in contrast to non-constitutively transcribed genes, which are only transcribed and translated by organisms in the face of a specific environmental stimuli and would thus result in lower druggability due to more limited gene expression.

3UND has been successfully expressed in E.coli (K-12) [4].

[|http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.55#Purification/COMMENTARY]
 * Purification Method:** It is possible to code for a 6 Histidine tag when engineering the BURPS1710b_3264 gene for transformation into an //E.coli// plasmid. The engineered N-terminus 6-His tag will then allow purification using Nickel-NTA column chromatography and FPLC. Other procedures, as documented on BRENDA, include “overnight dialysis against 50 mM ammonium acetate, pH 7.8, 4°C, reconstitution with substrates and products”


 * Image of protein:**


 * __Figure 4-__ PyMol surface image of 3UND tetramer with blue A subunit carbons, green B subunit carbons, pink C subunit carbons, and yellow D subunit carbons. Oxygen atoms are shown in red and nitrogen atoms in blue. **




 * __Figure 5__- PyMol cartoon of 3UND tetramer with blue A subunit, green B subunit, pink C subunit, and yellow D subunit. PEP shown as sticks colored by element with carbon as pink, oxygen as red, and phosphorous as orange. A5P shown as sticks colored by element with carbon as green, phosphate as orange, oxygen as red.**


 * __Figure 6__- A subunit of 3UND shown as ribbon with protein carbons shown as green. A5P is shown as sticks with yellow carbon, red oxygen, and orange phosphorous. PEP is shown as sticks with pink carbon, red oxygen, and orange phosphorous.**

MNLAGFEVGLDKPFFLIAGTCVVESEQMTIDTAGRLKEICAKLGVPFIYKSSYDKANRSS GKSFRGLGMDEGLRILAEVKRQLNVPVLTDVHEIDEIAPVAAVVDVLQTPAFLCRQTDFI RACAQSGKPVNIKKGQFLAPHDMKNVIDKARDAAREAGLSEDRFMACERGVSFGYNNLVS DMRSLAIMRETGAPVVFDATHSVQLPGGQGTSSGGQREFVPVLARAAVATGVAGLFMETH PNPAEAKSDGPNAVPLGRMAALLETLVTLDQAVKRVPFLENDFN
 * Amino Acid Sequence:**


 * Length of your protein in Amino Acids:** 281 AA per chain, 1124 AA per tetramer
 * Molecular weight of protein in kDa using Expasy ProtParam:** 30150.9 kDa per chain
 * Molar Extinction coefficient of protein at 280nm:** 4845 M-1cm-1 for A subunit


 * TMpred graph Image**:

__F**igure 7-**__ **TMpred Output of the “A” subunit of 3UND isolated from //Burkholderia pseudomallei//****. Two possible transmembrane regions were identified with scores of 542 and 861.** Inside to outside helices : 2 found from to score center 97 ( 99) 115 ( 115) 110 107 222 ( 222) 241 ( 238) 573 230

Outside to inside helices : 1 found from to score center 221 ( 221) 237 ( 237) 700 229




 * __Figure 8-__TMpred output for all four (A, B, C, and D) subunits of 3UND isolated from //Burkholderia pseudomallei// showed 8 possible transmembrane helices with significance scores ranging from 542-861, as outputted by TMpred, ExPASy.**

Inside to outside helices : 8 found

from to score center

8 ( 8) 27 ( 27) 861 17

226 ( 226) 244 ( 244) 542 236

290 ( 290) 312 ( 309) 904 300

511 ( 511) 529 ( 529) 542 521

575 ( 575) 597 ( 594) 904 585

796 ( 796) 814 ( 814) 542 806

860 ( 860) 882 ( 879) 904 870

1081 (1081)1099 (1099) 542 1091

Outside to inside helices : 8 found

from to score center

7 ( 7) 30 ( 25) 1148 17

223 ( 226) 244 ( 242) 672 234

292 ( 292) 315 ( 310) 1148 302

508 ( 511) 529 ( 527) 672 519

577 ( 577) 600 ( 595) 1148 587

793 ( 796) 814 ( 812) 672 804

862 ( 862) 885 ( 880) 1148 872

1078 (1081)1099 (1097) 672 1089

atgaacgtagcaatcagccccggcgtcacggccggcaacagcctgcctttcgtgctgttc
 * CDS gene Sequence:**

ggcgggatcaacgtgctcgagagtctcgacttcacgctcgacgtgtgcggcgaatacgtc

gcggtgacgcgcaagctcggcattccgttcgtgttcaaggcgtcgttcgacaaggcgaac

cgctcgtcgatccattcgtatcgcggcgtcgggctcgacgaaggcctgaagatcttcgcc

gaggtgaaggcgcgcttcggcgtgccggtgatcaccgatgtgcacgaagccgagcaggcg

gcgcccgtggccgaaatcgccgacgtgttgcaggtgcccgcgtttctcgcgcggcagacc

gatctcgtcgtcgcgatcgcgaaggccggcaagccggtgaacgtgaagaagccgcagttc

atgagccccacgcaattgaagcacgtggtgtcgaaatgcggcgaggtcggcaacgatcgc

gtgatgctgtgcgagcgcggcagttcgttcggctacgacaatctcgtcgtggacatgctc

ggcttccggcagatggccgagacgacgggcggttgcccggtgatcttcgacgtcacgcac

agcctgcagtgccgcgatccgctcggcgacgcgtcgggcggccggcgccggcaagtgctc

gatctcgcgcgcgcgggcatcgcggtcggcatcgcggggctctttctcgaggcgcacccc

gatcccgaccgcgcgcgctgcgatgggccgagcgcgctgccgttgcatcagctcgagggc

ttgctgtcgcagatgaaggcgatcgacgatctcgtcaagcgcatgccggcgctcgagatt

cgatga

*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): ** not needed now ** *GC% Content for gene (codon optimized): ** not needed now **
 * GC% Content for gene:** 66.1%; (559/846) bases

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): ** not needed now **
 * 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. **


 * References **

[1] Northfield, J.; Whitty, C.; MacPhee, I. B//urkholderia pseudomallei// infection, or melioidosis, and nephrotic syndrome. //Nephrol. Dial. Transplant.// 2002, 17 (1), 137-139. doi:10.1093/ndt/17.1.137

[2] Baugh, L; Gallagher, L.A.; Patrapuvich R.; Clifton M.C. Combining Functional and Structural genomics to Sample the Essential Burkholderia Structome. //PLoS ONE.// 2013,** 8(1), 10033-10046.

[3] http://www.ssgcid.org/target-status/featured-structures/kdop/

[4] http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/enzymes/GetPage.pl?ec_number=2.5.1.55