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Deciphering the Resistance Profile of Cancerous Thymidylate Synthase
Author(s) -
Islam Zahidul,
Gurevic Ilya,
Saeed Muhammad,
Rasool Sobia,
Kohen Am
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.921.12
Subject(s) - thymidylate synthase , mutant , dna , mutation , enzyme , biochemistry , biology , thymine , chemistry , microbiology and biotechnology , genetics , gene , cancer , fluorouracil
Thymidylate Synthase (TSase) is an enzyme that converts 2′‐deoxyuridinemonophosphate (dUMP) to thymidylate (dTMP), a base for DNA. Since there is no other de novo route to dTMP in cells, TSase is critical to cell replication and survival. 5F‐dUMP – produced intracellularly from the chemotherapeutic pro‐drug 5‐fluorouracil (5‐FU) – inhibits TSase, thus halting the supply of the DNA base thymidylate, causing cessation of DNA synthesis and leading to thymine‐less cell death. While it is very common for pathogenic organisms to become resistant to certain antibiotic drugs, cancerous cells have also developed some mechanisms to cope with the cytotoxic effect of chemotherapeutic drugs. 1 A variant of human ( hs ) TSase (Y33H) derived from a primary culture of human colonic tumor was reportedly found to confer resistance to 5‐FU.2 Crystal structures of hs TSase indicate Y33 is a remote residue that does not make direct contact either with the inhibitor or the residues interacting with it. Nevertheless, mutation at Y33 affects the affinity of the inhibitor for the enzyme. To resolve this enigma, we aim to investigate the molecular origin behind the resistance due to this remote mutation. Recombinant WT hs TSase and the mutant Y33H were expressed and purified in E. coli host systems. We structurally and kinetically interrogated WT and the mutant. Our studies revealed that the mutation at Y33 causes subtle changes across the protein that affect the chemical transformations at the active site. This change in protein could account for the low affinity of the mutant for the drug 5‐FdUMP.