1kzi
From Proteopedia
(New page: 200px<br /><applet load="1kzi" size="450" color="white" frame="true" align="right" spinBox="true" caption="1kzi, resolution 1.75Å" /> '''Crystal Structure of...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1kzi.gif|left|200px]]<br /><applet load="1kzi" size=" | + | [[Image:1kzi.gif|left|200px]]<br /><applet load="1kzi" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1kzi, resolution 1.75Å" /> | caption="1kzi, resolution 1.75Å" /> | ||
'''Crystal Structure of EcTS/dUMP/THF Complex'''<br /> | '''Crystal Structure of EcTS/dUMP/THF Complex'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Mutant forms of thymidylate synthase (TS) with substitutions at the | + | Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer. |
==About this Structure== | ==About this Structure== | ||
| - | 1KZI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CO3, UMP, THG, DTU, DTT and GOL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Thymidylate_synthase Thymidylate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.45 2.1.1.45] Full crystallographic information is available from [http:// | + | 1KZI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CO3:'>CO3</scene>, <scene name='pdbligand=UMP:'>UMP</scene>, <scene name='pdbligand=THG:'>THG</scene>, <scene name='pdbligand=DTU:'>DTU</scene>, <scene name='pdbligand=DTT:'>DTT</scene> and <scene name='pdbligand=GOL:'>GOL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Thymidylate_synthase Thymidylate synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.45 2.1.1.45] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KZI OCA]. |
==Reference== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Thymidylate synthase]] | [[Category: Thymidylate synthase]] | ||
| - | [[Category: Finer-Moore, J | + | [[Category: Finer-Moore, J S.]] |
| - | [[Category: Fritz, T | + | [[Category: Fritz, T A.]] |
[[Category: Liu, L.]] | [[Category: Liu, L.]] | ||
| - | [[Category: Stroud, R | + | [[Category: Stroud, R M.]] |
[[Category: CO3]] | [[Category: CO3]] | ||
[[Category: DTT]] | [[Category: DTT]] | ||
| Line 26: | Line 26: | ||
[[Category: enzyme substrate complex]] | [[Category: enzyme substrate complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:39:36 2008'' |
Revision as of 11:39, 21 February 2008
|
Crystal Structure of EcTS/dUMP/THF Complex
Overview
Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.
About this Structure
1KZI is a Single protein structure of sequence from Escherichia coli with , , , , and as ligands. Active as Thymidylate synthase, with EC number 2.1.1.45 Full crystallographic information is available from OCA.
Reference
Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access., Fritz TA, Liu L, Finer-Moore JS, Stroud RM, Biochemistry. 2002 Jun 4;41(22):7021-9. PMID:12033935
Page seeded by OCA on Thu Feb 21 13:39:36 2008
Categories: Escherichia coli | Single protein | Thymidylate synthase | Finer-Moore, J S. | Fritz, T A. | Liu, L. | Stroud, R M. | CO3 | DTT | DTU | GOL | THG | UMP | Enzyme substrate complex
