Artificial duplication of the R67 dihydrofolate reductase gene to create protein asymmetry. Effects on protein activity and folding.


Abstract

R67 dihydrofolate reductase (DHFR), encoded by an R plasmid, provides resistance to the antibacterial drug trimethoprim. This enzyme does not exhibit any structural or sequence homologies with chromosomal DHFR. A recent crystal structure of tetrameric R67 DHFR (D. Matthews, X. Nguyen-huu, and N. Narayana, personal communication) shows a single pore traversing the length of the molecule. Numerous physical and kinetic experiments suggest the pore is the active site. Since the center of the pore possesses exact 222 symmetry, mutagenesis of residues designed to explore substrate binding will probably also affect cofactor binding. As a first step in breaking this inevitable symmetry in R67 DHFR, the gene has been duplicated. The protein product, R67 DHFRdouble, is twice the molecular mass of native R67 DHFR and is fully active with kcat = 1.2 s-1, Km(NADPH) = 2.7 microM and Km(dihydrofolate) = 6.3 microM. Equilibrium unfolding studies in guanidine-HCl indicate R67 DHFRdouble is more stable than native R67 DHFR at physically reasonable protein concentrations. Microcalorimetry studies show native R67 DHFR undergoes fully reversible thermal unfolding. Unfolding can be described by a two-state process since a ratio of delta Hcalorimetric to delta Hvan't Hoff equals 0.96. In contrast, thermal unfolding of R67 DHFRdouble is not fully reversible and possesses an oligomerization component introduced by the gene duplication event. Study holds ProTherm entries: 5233 Extra Details: active site; substrate binding; two-state process;,oligomerization component

Submission Details

ID: tKj6TnQD3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Zhuang P;Yin M;Holland JC;Peterson CB;Howell EE,J. Biol. Chem. (1993) Artificial duplication of the R67 dihydrofolate reductase gene to create protein asymmetry. Effects on protein activity and folding. PMID:8226776
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2RH2 2008-06-03 0.96 High Resolution DHFR R-67
2GQV 2007-04-24 1.1 High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site
2P4T 2007-06-05 1.15 Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode
2RK1 2008-06-03 1.26 DHFR R67 Complexed with NADP and dihydrofolate
6NY0 2019-05-29 1.4 Crystal structure of trimethoprim-resistant type II dihydrofolate reductase in complex with a bisbenzimidazole inhibitor
3SFM 2011-11-02 1.4 Novel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure
1VIE 1997-10-22 1.7 STRUCTURE OF DIHYDROFOLATE REDUCTASE
6NXZ 2019-05-29 1.75 Crystal structure of trimethoprim-resistant type II dihydrofolate reductase in complex with a bisbenzimidazole inhibitor
1VIF 1997-10-22 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE
2RK2 2008-06-03 1.9 DHFR R-67 complexed with NADP

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase type 2 P00383 DYR21_ECOLX