TargetF15+-+Verona-integron+encoded+Metallo-beta+Lactamase+(Pseudomonas+aeruginosa)

**Target:** Verona-integron encoded Beta-metallo lactamase (VIM-2)


 * Gene ID: **14678525
 * Protein ID: **YP_007509601

**Organism:** Pseudomonas aeruginosa
 * Etiological Risk Group: **Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia

Pseudomonas aeruginosa is a bacterium that causes a wide variety of infections in hospital patients and those with impaired immune systems. Infections by this organism are treated with antibiotics; however, with increasing bacterial resistance to antibiotics, this becomes more difficult. In particular, infections by multidrug-resistant strains of P. aeruginosa can be a serious issue as it extremely difficult to find antibiotics that can inhibit the bacteria. The CDC estimates that there are approximately 51,000 cases of infection by P. aeruginosa in the healthcare setting per year with 6,000 of these cases by multidrug-resistant strains. Of the various ways that bacteria can develop resistance to antibiotics, the most common is the development of an enzyme that can neutralize the drug. In P. aeruginosa, this enzyme is VIM-2, a metallo-beta-lactamase that hydrolyzes the beta-lactam ring in carbapanems to neutralize their effectiveness [1]. As this enzyme is a Class B beta-lactamase, it is able to hydrolyze nearly all beta-lactam antibiotics [2]. This enzyme is also viable for horizontal gene transfer across different bacteria, augmenting the rate at which the bacteria can become multidrug-resistant. Because of the threat that VIM-2 and other metallo-beta-lactamases poses to hospitals and other medical institutions,
 * Disease: **


 * TDR_Targets:** None
 * GeneDatabase:** http://www.ncbi.nlm.nih.gov/gene/14678525

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC89788/ BindingDB shows one current inhibitor with Ki value: http://bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?tag=pol&submit=Search&target=beta-lactamase%20vim-2&polymerid=50004301
 * Essentiality:** Essential for neutralizing carbapenams and other beta-lactamases.
 * Druggability:**

**E.C.:** 3.5.2.6 **Structure:** 1 polymer, 1 or 2 chains **PDB:** 1KO2, 1KO3, 4BZ3, 4C1D, 4C1E, 4NQ2, 4PVO, 4PVT, 4UA4



http://www.brenda-enzymes.org/enzyme.php?ecno=3.5.2.6&Suchword=&organism%5B%5D=Pseudomonas+aeruginosa&show_tm=0
 * Reaction:** Beta-lactam + water -> substituted beta-amino acid

DH5a E. coli, pNIC-Bsa4 vector, Kanamycin and Sucrose selection system 6 positive clones out of 8 tested colonies. ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAA CGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGA GATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCG TCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTG CTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCT GGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTT GTAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAA CGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGA GATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCG TCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTG CTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCT GGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTT GTAAAAGCGCACAC N AATCGCTCAGTCGTTGAGTAG ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTAT T GACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAAC GCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAG ATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCGT CGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTGC TGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTG GCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTG TAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG
 * Cloning:**
 * Results:**
 * Colonies 1, 2, 4, 5, 6, 8 **
 * Colony 3 **
 * Colony 7 **

BL21(DE3) E. coli, induced by IPTG Successful transformation into BL21(DE3) cells.
 * Expression:**
 * Results:**

6x His Tag, Ni-NTA column purification with imidazole FPLC (gel here)
 * Purification:**
 * Results:**

Colorimetric assay using Nitrocefin as substrate. Nitrocefin http://www.toku-e.com/product/nitrocefin/
 * Enzyme Assay:**
 * Substrate:**


 * Current Inhibitors:**

Sodium 2,3-dimethylmaleate

Met S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E M H H H H H H S S G V D L G T E N L Y F Q S M S P L A F S V D S S G E Y P T V S E I P V G E V R L Y Q I A D G V W S H I A T Q S F D G A V Y P S N G L I V R D G D E L L L I D T A W G A K N T A A L L A E I E K Q I G L P V T R A V S T H F H D D R V G G V D V L R A A G V A T Y A S P S T R R L A E V E G N E I P T H S L E G L S S S G D A V R F G P V E L F Y P G A A H S T D N L V V Y V P S A S V L Y G G C A I Y E L S R T S A G N V A D A D L A E W P T S I E R I Q Q H Y P E A Q F V I P G H G L P G G L D L L K H T T N V V K A H T N R S V V E
 * Amino Acid Sequence:**
 * Length:**
 * Amino Acid Sequence with His Tag:**
 * Length:** 269 aa
 * Weight:** 28.802 kDa
 * Molar Extinction Coefficient:** 31400
 * pI:** 5.15

http://web.expasy.org/cgi-bin/protparam/protparam
 * ProtParam:**


 * TmPred:**

ATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCGGGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATG GCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAAC GCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGGGTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACG CACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATCCTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTG GTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATGGCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGG CCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCAGTCGTTGAGTAG ATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGAGTCCGCTCGCTTTTTCCGTAGATTCTAGCGGTGAGTATCCGACAGTCAGCGAAATTCCGGTCG GGGAGGTCCGGCTTTACCAGATTGCCGATGGTGTTTGGTCGCATATCGCAACGCAGTCGTTTGATGGCGCAGTCTACCCGTCCAATGGTCTCATTGTCCGTGATGGTGATGAGTTGCTTTTGATTGATACAGC GTGGGGTGCGAAAAACACAGCGGCACTTCTCGCGGAGATTGAGAAGCAAATTGGACTTCCTGTAACGCGTGCAGTCTCCACGCACTTTCATGACGACCGCGTCGGCGGCGTTGATGTCCTTCGGGCGGCTGGG GTGGCAACGTACGCATCACCGTCGACACGCCGGCTAGCCGAGGTAGAGGGGAACGAGATTCCCACGCACTCTCTAGAAGGACTCTCATCGAGCGGGGACGCAGTGCGCTTCGGTCCAGTAGAACTCTTCTATC CTGGTGCTGCGCATTCGACCGACAACTTAGTTGTGTACGTCCCGTCTGCGAGTGTGCTCTATGGTGGTTGTGCGATTTATGAGTTGTCACGCACGTCTGCGGGGAACGTGGCCGATGCCGATCTGGCTGAATG GCCCACCTCCATTGAGCGGATTCAACAACACTACCCGGAAGCACAGTTCGTCATTCCGGGGCACGGCCTGCCGGGCGGTCTAGACTTGCTCAAGCACACAACGAATGTTGTAAAAGCGCACACAAATCGCTCA GTCGTTGAGTAG
 * DNA Sequence:**
 * Sequence with His Tag:**
 * Length:** 744 bp
 * GC Content:** 56.048387%
 * CDS Codon Optimized:** N/A
 * Overlap Primers:** N/A

Forward: 5’ TACTTCCAATCC ATGAGTCCGCTCGCTTTTTCC 3’ Reverse: 5’ TATCCACCTTTACTG CTACTCAACGACTGAGCGAT 3’
 * Primers (pLIC Cloning):**