Target-dUTP+diphosphatase+(Leishmania+major)


 * Leishmania major **


 * *Target (protein/gene name): **** deoxyuridine triphosphatase **


 * NCBI Gene # or RefSeq#: ** **13391737**

**REFSEQ**# : 157864228


 * *Protein ID (NP or XP #) or Wolbachia#: **** 29483 **


 * *Organism: **** Leishmania major **


 * *Background/Disease Information: **The parasite is an intracellular pathogen of the immune system targeting macrophages and dendritic cells. The disease Leishmaniasis affects the populations of 88 counties worldwide with symptoms ranging from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes to visceral disease affecting the haemopoetic organs.


 * Essentiality of this protein: ****Gene/Ortholog:** Tb927.7.5160 (OG4_25847); **Phenotype:** significant loss of fitness in differentiation of procyclic to bloodstream forms; **Source study:** alsford

The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding.

removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA, essential for replication and cell survival


 * *EC#: ** 3.6.1.23

**Link to BRENDA EC# page: []**

Figure 1: Reaction by dUTP diphosphatase


 * Enzyme Assay information **

[] ITC assay - don't have in VDS lab. Or pyrophosphate - less easy vs. phosphate. --Dr. B
 * -- link to paper that contains assay information **


 * -- List cost and quantity of substrate reagents and supplier **
 * GenScript: $282.45 Full Length Sequence **


 * -- PDB # or closest PDB entry if using homology model: 2YAY **


 * Current Inhibitors: Calcium and DUP **


 * Image of protein (PyMol or etc): **

Figure 2: Image of dUTP from PyMOL.


 * *Amino Acid Sequence: **

MKRARSANIP GAILHSLAEL QDGLNAMIDP SWRAVRSLDN WALAITMEST ELLDSYPWKW

WKNLNATPDL ANVRIELVDI FHFSLSGAMQ MRSTPDDEIP AASLKPLKEV MTTFLPAKEC

TSDPYGFVFF PLTDTQNAIA SFRNIIQLAN AYRFDVIIEC IIYAAEDLGF NLVAYYIAKH

TLNCIRQLSG YKDGSYVKVN NGVEDNSLLH NCIKDVSLDE VLDADKYVQA WNSIMANVYE

AFQIKESDRK DAERWFALAK ENRLAIKA

The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates.
 * Purification**:

=** *length of your protein in Amino Acids: 268 **=

**Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6**


 * Molar Extinction coefficient of your protein at 280 nm wavelength: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. **


 * Ext. coefficient 53650 **
 * Abs 0.1% (=1 g/l) 1.768, assuming all pairs of Cys residues form cystines **

code
 * Ext. coefficient 53400 **
 * Abs 0.1% (=1 g/l) 1.759, assuming all Cys residues are reduced **

code


 * *CDS Gene Sequence **

1 ATGAAACGTGCGCGTAGCGCGAACATCCCGGGTGCCATCCTGCACTCTCTCGCGGAGCTG

61 CAGGACGGTCTCAACGCGATGATCGACCCGTCTTGGCGTGCGGTTCGTTCTCTCGACAAC

121 TGGGCCCTCGCGATCACCATGGAATCTACCGAACTGCTCGACTCCTACCCGTGGAAGTGG

181 TGGAAGAATCTGAACGCTACCCCGGACCTGGCCAATGTGCGTATCGAACTGGTTGACATC

241 TTCCATTTCTCTCTGTCTGGTGCGATGCAGATGCGTAGCACTCCGGACGATGAAATTCCA

301 GCCGCGTCCCTGAAACCGCTGAAAGAAGTTATGACCACCTTCCTGCCGGCGAAAGAATGC

361 ACCTCTGACCCGTATGGTTTCGTTTTTTTCCCGCTCACCGACACCCAGAACGCAATCGCA

421 TCTTTCCGTAATATCATCCAGCTCGCGAATGCCTACCGTTTCGACGTTATCATCGAGTGC

481 ATCATCTACGCAGCTGAAGACCTGGGTTTCAACCTGGTTGCGTACTACATCGCGAAACAC

541 ACCCTGAACTGCATCCGTCAGCTCTCTGGTTACAAAGACGGTTCTTACGTTAAAGTTAAC

601 AACGGTGTTGAAGACAACTCTCTGCTGCACAATTGCATTAAAGATGTTTCTCTGGACGAG

661 GTCCTCGACGCGGACAAATACGTTCAGGCGTGGAACTCTATCATGGCGAACGTCTACGAA

721 GCGTTCCAGATCAAAGAGTCTGACCGTAAAGACGCAGAACGTTGGTTTGCGCTGGCGAAG

781 GAAAACCGCCTGGCGATCAAGGCGTAA

Figure 3: TMpred output for dUTP indicating the protein as not a membrane bound protein.