PMID: 9543003May 23, 1998Paper

A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition

Protein Engineering
H ParkE E Howell

Abstract

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to increasing clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a single active site pore that spans the length of the homotetramer. Related sites (due to a 222 symmetry element at the center of the pore) are used to bind ligands, i.e. each half of the pore can accommodate either the substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and NADPH are bound differently. To evaluate the role of glutamine 67 (and its symmetry-related Q167, Q267 and Q367 residues which occur at the center of the active site pore), a Q67H mutation was constructed. Binary binding of dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays two identical sites with a Kd value of 0.04 microM, while binding of NADPH shows two sites possessing negative cooperativity with Kd values of 0.027 and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type (wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and DHF binding in Q67H R67 DHFR no longer displays positive coop...Continue Reading

Citations

Aug 2, 2005·Journal of Computer-aided Molecular Design·Hernán AlonsoJill E Gready
Jul 24, 2002·International Journal of Antimicrobial Agents·F M E WagenlehnerK G Naber
Dec 5, 2015·Biochemistry·Michael R DuffElizabeth E Howell
Dec 7, 2007·The Urologic Clinics of North America·F M E WagenlehnerK G Naber
May 3, 2007·Protein Science : a Publication of the Protein Society·N DivyaNarendra Narayana
Nov 18, 2014·Protein Science : a Publication of the Protein Society·Maximilian C C J C EbertJoelle N Pelletier
Apr 6, 2006·Trends in Microbiology·Hernán Alonso, Jill E Gready
Jun 7, 2005·Expert Opinion on Emerging Drugs·Florian M E WagenlehnerKurt G Naber
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
Apr 30, 2008·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Atsushi YahashiriAmnon Kohen
Aug 31, 2004·The Journal of Biological Chemistry·Stephanie N HicksElizabeth E Howell

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