Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein.


Abstract

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli. Study holds ProTherm entries: 4022, 4023 Extra Details: antibacterial drug trimethoprim; beta-barrel structure;,gene coding; two-state model

Submission Details

ID: cfFsSEDG4

Submitter: Connie Wang

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

Version: 1

Publication Details
Reece LJ;Nichols R;Ogden RC;Howell EE,Biochemistry (1991) Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. PMID:1932013
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