Target+-+GES-5+carbapenemase+(K.+pneumoniae)

**Etiologic Risk Group (see link below):** RG2
 * Target (protein/gene name):** GES-5 carbapenemase (blaGES-5)
 * NCBI Gene # or RefSeq#:** 9389494
 * Protein ID (NP or XP #) or Wolbachia#:** (none available on PubChem)
 * Organism (including strain):** //Klebsiella pneumoniae//

//Klebsiella pneumoniae// causes a type of pneumonia that is associated with destructive changes in the lungs. It has a rapid onset and often-fatal outcome despite early and appropriate antimicrobial treatment. Patients with this type of penumonia typically experience an acute onset of high fever and chills, flulike symptoms, and productive cough with abundant, thick, blood-tinged sputum (1). //Klebsiella// bacteria can be spread from person-to-person contact and less commonly by contamination of the environment (not spread through air). From contact, //Klebsiella// bacteria may enter the respiratory tract or bloodstream. Patients whose care requires devices like ventilators, intravenous catheters and patients who take long courses of certain antibiotics are most at risk (2) //Klebsiella b//acteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems (2). This is because //Klebsiella pneumoniae// are beginning to make carbapenemases, enzymes that break down carbapenems. making them ineffective. One report cites that they carbapenemases can contribute to death in up to 50% of patients who become infected. Carbapenemase-producing //K. pneumoniae// was very uncommon in the United States before 1992 and the first report on its prevalence in the US was not reported until 2001 (3). Carbapenemase-producing bacteria have also emerged in multiple species of gram-negative bacteria across the world (4).
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up): **

(1) http://emedicine.medscape.com/article/219907-clinical (2) http://www.cdc.gov/HAI/organisms/klebsiella/klebsiella.html#a5 (3) http://www.cdc.gov/hai/organisms/cre/index.html (4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075864/

**Link to TDR Targets page (if present):** None available **Essentiality of this protein:** Yes "Acquired resistance to broad-spectrum β-lactams is increasingly observed in P. aeruginosa. Currently, PER-, VEB-, and GES-type enzymes are the most frequently observed extended-spectrum β-lactamases (ESBLs) identified in Pseudomonas spp. Therefore, carbapenems are considered crucial for treating many P. aeruginosa-associated infections." __Article:__ Rapid Detection of Carbapenemase-Producing Pseudomonas spp. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486216/) **Complex of proteins:**No but commonly produced as a dimer (can still function as a monomer) "The 28-day mortality was 13.3% in the combination therapy group compared with 57.8% in the monotherapy group (P = 0.01). The most commonly used combinations were colistin-polymyxin B or tigecycline combined with a carbapenem … The use of combination therapy for definitive therapy appears to be associated with improved survival in bacteremia due to KPC-producing K. pneumoniae." __Article:__ Treatment Outcome of Bacteremia Due to KPC-Producing Klebsiella pneumoniae: Superiority of Combination Antimicrobial Regimens (http://aac.asm.org/content/56/4/2108.abstract)
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): ** http://www.ncbi.nlm.nih.gov/gene/9389494
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **

Clavulanic Acid __Article:__ University of Notre Dame: Importance of position 170 in the inhibition of GES-type Beta-lacatmases by clavulanic acid (http://books.google.com/books?id=SKOcDOCZN_UC&pg=PA14&lpg=PA14&dq=potential+inhibitors+for+GES-5+carbapenemase&source=bl&ots=SZDECjfoPD&sig=YZtaTUXJ6Z0UoDU9URirksAd1Jw&hl=en&sa=X&ei=jDZgU4jPJMOGyASG7YHoAQ&ved=0CHMQ6AEwCQ#v=onepage&q=potential%20inhibitors%20for%20GES-5%20carbapenemase&f=false)


 * EC#: ** 3.5.2.6
 * Link to BRENDA EC# page:** http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6

Penicillinase Benzylpenicillin as Substrate (commonly known as Penicillin G) __Method:__ Titrimetric __Cost:__ $17.00 __Quantity__: 356.37g __Supplier__: BioReagent __CAS#__: 69-57-8
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**

__Link for Procedure:__ http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/penicillbenzyl.pdf __Link for MSDS sheet:__ http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=P3032&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fsearch%3Finterface%3DAll%26term%3Dpenicillin%2Bg%26N%3D0%26mode%3Dmatch%2520partialmax%26focus%3Dproduct%26lang%3Den%26region%3DUS

__**Structure Available (PDB or Homology model)**__ __PDB#:__ 4H8R __Pairwise alignment of your BLASTP search in NCBI against the PDB __ __Query Coverage__: 26-129 __Max % Identities__: 33/104 (32%) __% Positives__: 43/104 (41%) __Chain used for homology__: Chain A

β-Lactamase purification. Cultures of //E. coli// TOP10 harboring recombinant plasmid pTOPO-GES-14 were grown overnight at 37°C in 4 liters of TS broth containing amoxicillin (50 μg/ml) and kanamycin (30 μg/ml). β-Lactamase GES-14 was purified by ion-exchange chromatography. Briefly, the bacterial suspension was pelleted, resuspended in 40 ml of 100 mM sodium phosphate buffer (pH 7.0), sonicated, cleared by ultracentrifugation, and treated with DNase. The extract was then dialyzed against 20 mM bis-Tris {[bis(2-hydroxyethyl-imino]tris(hydroxymethyl)methane}(pH 6.5) and loaded onto a preequilibrated Q-Sepharose column with the same buffer. The resulting enzyme extract was recovered in the flowthrough and dialyzed against 20 mM Tris-HCl (pH 7.5) overnight at 4°C. This extract was then loaded onto a preequilibrated (20 mM Tris-HCl [pH 7.5]) Q-Sepharose column, and the proteins were eluted with a linear NaCl gradient (0 to 0.5 M). The β-lactamase activity was eluted with NaCl at a concentration of 200 mM in the same Tris-HCl buffer. Finally, fractions containing the highest β-lactamase activities were pooled and subsequently dialyzed overnight against 100 mM phosphate buffer (pH 7.0). The β-lactamase activity was determined qualitatively using nitrocefin hydrolysis (Oxoid, Dardilly, France). The protein content was measured using the Bio-Rad DC protein assay. __Article:__ Carbapenem-Hydrolyzing GES-Type Extended-Spectrum β-Lactamase in //Acinetobacter baumannii (//http://aac.asm.org/content/55/1/349.long)
 * Current Inhibitors:** dCHEMBL777, TAZOBACTUM, and CHEMBL403
 * Expression Information (has it been expressed in bacterial cells):** Yes
 * Purification Method: **
 * Image of protein (PyMol** **with features delineated and shown separately):**

MRFIHALLLAGIAHSAYASEKLTFKTDLEKLEREKAAQIGVAIVDPQGEIVAGHRMAQRFAMCSTFKFPLAALVFERIDSGTERGDRKLSYGPDMIVEWSPATERFLASGHMTVLEAA QAAVQLSDNGATNLLLREIGGPAAMTQYFRKIGDSVSRLDRKEPEMSDNTPGDLRDTTTPIAMARTVAKVLYGGALTSTSTHTIERWLIGNQTGDATLRAGFPKDWVVGEKTGTCAN GGRNDIGFFKAQERDYAVAVYTTAPKLSAVERDELVASVGQVITQLILSTDK
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

25565 Abs 0.1% (=1 g/l) 0.820, assuming all pairs of Cys residues form cystines 25440 Abs 0.1% (=1 g/l) 0.816, assuming all Cys residues are reduced
 * Length of your protein in Amino Acids:** 287 residues
 * Molecular Weight of your protein:** 31184.5 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**


 * TMpred graph Image **:

ATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGA CTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCC GCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAACAGG CATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGC TCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACA AAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGA CTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGG CGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCG GCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAA **GC% Content for gene:** 61.5%
 * CDS Gene Sequence (paste as text only):**

CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):

GC% Content for gene (codon optimized):

Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):

Primer design results for 'tail' primers (this is just 2 sequences): 
 * Enzymatic Assay Option 2 || Penicillinase Cephaloridine as Substrate ||
 * Enzymatic Assay Option 2 Method || Continuous Spectrophotometric Rate Determination ||