Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations.

The Journal of Biological Chemistry
Stephanie N HicksE E Howell

Abstract

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1+2 and K32M: 1+4, which place the mutations on opposite sides of the pore, r...Continue Reading

References

Dec 2, 1975·Biochemistry·D BaccanariJ Burchall
Nov 4, 1976·Journal of Molecular Biology·M T RecordP De Haseth
Feb 12, 1991·Biochemistry·R M BritoP R Rosevear
Jan 15, 1987·The Biochemical Journal·G M Umezurike
Oct 25, 1982·Journal of Molecular Biology·I D KuntzT E Ferrin
Jan 1, 1982·Methods in Enzymology·K J Ellis, J F Morrison
Sep 1, 1993·Protein Engineering·B K Shoichet, I D Kuntz
Jan 1, 1995·Methods in Enzymology·R W Woody
Nov 1, 1993·Protein Science : a Publication of the Protein Society·C A RoyerC R Matthews
Mar 26, 1998·Protein Science : a Publication of the Protein Society·L T ChongB Tidor
Oct 3, 1999·Methods in Enzymology·E De Bernardez ClarkR Rudolph
Mar 29, 2000·Biochemistry·F W WestE E Howell
Nov 15, 2001·Journal of the American Chemical Society·C Park, R T Raines
May 1, 2002·Chemistry : a European Journal·Sjoerd HarderTimo Repo
Sep 10, 2003·Biochemistry·Stephanie N HicksElizabeth E Howell
Sep 23, 2003·Biophysical Chemistry·Ralph C Dougherty, Louis N Howard

❮ Previous
Next ❯

Citations

Aug 2, 2005·Journal of Computer-aided Molecular Design·Hernán AlonsoJill E Gready
Nov 17, 2011·PLoS Biology·Arvind Ramanathan, Pratul K Agarwal
Dec 5, 2015·Biochemistry·Michael R DuffElizabeth E Howell
Apr 6, 2006·Trends in Microbiology·Hernán Alonso, Jill E Gready
Apr 7, 2005·Chembiochem : a European Journal of Chemical Biology·Elizabeth E Howell
Dec 4, 2004·Protein Engineering, Design & Selection : PEDS·Andreea R SchmitzerJoelle N Pelletier
May 24, 2006·Biochemistry·Shaileja ChopraElizabeth E Howell
Aug 28, 2010·Biochemistry·Ganesh KamathPratul K Agarwal
Sep 15, 2005·Biochemistry·Michael JacksonElizabeth E Howell

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.