1qqq
From Proteopedia
| Line 1: | Line 1: | ||
[[Image:1qqq.gif|left|200px]] | [[Image:1qqq.gif|left|200px]] | ||
| - | + | <!-- | |
| - | + | The line below this paragraph, containing "STRUCTURE_1qqq", creates the "Structure Box" on the page. | |
| - | + | You may change the PDB parameter (which sets the PDB file loaded into the applet) | |
| - | + | or the SCENE parameter (which sets the initial scene displayed when the page is loaded), | |
| - | + | or leave the SCENE parameter empty for the default display. | |
| - | | | + | --> |
| - | | | + | {{STRUCTURE_1qqq| PDB=1qqq | SCENE= }} |
| - | + | ||
| - | + | ||
| - | }} | + | |
'''CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE''' | '''CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE''' | ||
| Line 36: | Line 33: | ||
[[Category: Shaw, D.]] | [[Category: Shaw, D.]] | ||
[[Category: Spencer, H T.]] | [[Category: Spencer, H T.]] | ||
| - | [[Category: | + | [[Category: Methyltransferase]] |
| - | [[Category: | + | [[Category: Thymidylate synthase]] |
| - | [[Category: | + | [[Category: Transferase]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 06:35:43 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 03:35, 3 May 2008
CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE
Overview
Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.
About this Structure
1QQQ is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254., Fantz C, Shaw D, Jennings W, Forsthoefel A, Kitchens M, Phan J, Minor W, Lebioda L, Berger FG, Spencer HT, Mol Pharmacol. 2000 Feb;57(2):359-66. PMID:10648646 Page seeded by OCA on Sat May 3 06:35:43 2008
