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Novel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structureNovel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure
Structural highlights
Function[DYR21_ECOLX] Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Publication Abstract from PubMedTrimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF. Using a modified crystallization protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystallized and the structure was solved at 1.4 A resolution. Surprisingly, only wild-type protomers were incorporated into the crystal. Further experiments demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations. Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure.,Yachnin BJ, Colin DY, Volpato JP, Ebert M, Pelletier JN, Berghuis AM Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Nov 1;67(Pt, 11):1316-22. Epub 2011 Oct 25. PMID:22102224[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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