1bjg
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(New page: 200px<br /><applet load="1bjg" size="450" color="white" frame="true" align="right" spinBox="true" caption="1bjg, resolution 2.3Å" /> '''D221(169)N MUTANT DOE...)
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Revision as of 09:34, 20 November 2007
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D221(169)N MUTANT DOES NOT PROMOTE OPENING OF THE COFACTOR IMIDAZOLIDINE RING
Overview
In thymidylate synthase (TS), the invariant residue Asp-221 provides the, only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except, cysteine abolish activity. We have determined the crystal structures of, two ternary complexes of the Escherichia coli mutant D221N. In a complex, with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP, is covalently bound to the active site cysteine, as usual. CB3717, which, has no imidazolidine ring, is also bound in the usual productive, orientation, but is less ordered than in wild-type complexes. The side, chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of, CB3717, which must be in the enol form. In contrast, the structure of, D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate, shows the cofactor bound in two partially occupied, nonproductive binding, sites. In both binding modes, the cofactor has a closed imidazolidine ring, and adopts the solution conformation of the unbound cofactor. In one of, the binding sites, the pterin ring is turned around such that Asn-221, hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the, cofactor. This orientation blocks the conformational change required for, forming covalent ternary complexes. Taken together, the two crystal, structures suggest that the hydrogen bond between the side chain of, Asp-221 and N3 of the cofactor is most critical during the early steps of, cofactor binding, where it enforces the correct orientation of the pterin, ring. Proper orientation of the cofactor appears to be a prerequisite for, opening the imidazolidine ring prior to formation of the covalent, steady-state intermediate in catalysis.
About this Structure
1BJG is a Single protein structure of sequence from Escherichia coli with UFP and TMF as ligands. Active as Thymidylate synthase, with EC number 2.1.1.45 Full crystallographic information is available from OCA.
Reference
D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine., Sage CR, Michelitsch MD, Stout TJ, Biermann D, Nissen R, Finer-Moore J, Stroud RM, Biochemistry. 1998 Sep 29;37(39):13893-901. PMID:9753479
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