ISSRNS 2012: Abstracts / Synchrotron Radiation in Natural Science Vol. 11, No 1 – 2 (2012) P 21
CRYSTAL STRUCTURES OF MOUSE THYMIDYLATE SYNTHASE IN BINARY COMPLEX WITH A STRONG INHIBITOR, N(4)-OH-dCMP,
AND TERNARY COMPLEX WITH N(4)-OH-dCMP AND THE COFACTOR PRODUCT, DIHYDROFOLATE
A. Jarmu la1∗, A. Dowiercia l1, P. Wilk1, W.R. Rypniewski2, B. Kierdaszuk3, and W. Rode1
1Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Str., 03–092 Warszawa, Poland
2Institute of Bioorganic Chemistry, Polish Academy of Sciences, 12–14 Noskowskiego Str., 61–704 Pozna´n, Poland
3Institute of Experimental Physics, Warsaw University, 93 ˙Zwirki i Wigury Str., 02–089 Warszawa, Poland Keywords: synchrotron radiation, inhibitor, thymidylate synthase, enzyme-ligand-cofactor complex
∗e-mail : a.jarmula@nencki.gov.pl
Thymidylate synthase (TS; EC 2.1.1.45), an en- zyme serving as a target in chemotherapy, catalyzes the conversion of 2’-deoxyuridine-5’-monophosphate (dUMP) to 2’-deoxythymidine-5’-monophosphate (dTMP), involving the reductive methylation of N(5,10)-methylenetetrahydrofolate (mTHF), func- tioning as both methyl donor and reducing agent.
N(4)-OH-dCMP is a substrate analogue, be- ing a potent mTHF-dependent, thus mechanism- based, slow-binding inhibitor of TS (Ki ∼ 50 nM).
Incubated with the enzyme and the cofactor, N(4)- OH-dCMP was shown to form a ternary complex, similar to the classical TS inhibitor, FdUMP. How- ever, when N(4)-OH-[5-3H]dCMP replaced dUMP in the reaction mixture, 3H abstraction from the uracil C(5) atom was not apparent, suggesting, the reaction to be inhibited at an earlier stage than with FdUMP. In solution the equilibrium between rotamers around the C(4) = N(4) bond is signifi- cantly shifted toward the syn rotamer, but surpris- ingly only the anti-imino form, with the -OH group on the side of C(5), appeared to be the active in- hibitor form.
In order to learn more about the inhibition mechanism, X-ray crystallographic studies of TS complexes with N(4)-OH-dCMP were undertaken.
Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N(4)-OH-dCMP (measured to 1.75 ˚A resolu- tion) or both N(4)-OH-dCMP and mTHF (two crys- tals measured to resolutions of 1.35 ˚A and 1.17 ˚A).
The structure of the mTS-N(4)-OH-dCMP com- plex was found to be closely similar to the cor- responding structure of the native complex, mTS- dUMP. The both other structures showed the en- zyme to be involved in a ternary complex with N(4)-OH-dCMP and non-covalently bound dihydro- folate (DHF), instead of expected mTHF, suggest- ing the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of
the one-carbon group to an as yet unknown site and reduction of mTHF to DHF. The structures showed no indication of proton release from the C(5) inhibitor atom. Instead, both the C(5) and C(6) atoms showed an sp3 hybridization, suggesting re- duction and subsequent presence of second proton at C(5). In accord with our previous results, in all three complexes the molecule of N(4)-OH-dCMP was found in the anti rotameric form, with the latter probably playing a role in the mechanism of ligand recognition by TS.
Figure 1 : Superimposition of the subunits A of the crys- tal structures of the complexes between mTS and N(4)- OH-dCMP (green), and mTS and dUMP (light blue).
The atoms of nitrogen, oxygen and phosphorus in the ligands are colored blue, red and orange, respectively.
Acknowledgments: Supported by the National Sci- ence Centre (grant No. 2011/01/B/NZ6/01781) and the Ministry of Science and Higher Education (grant No.
N301 3948 33).
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