Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase.


Abstract

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction. Study holds ProTherm entries: 2896, 2897, 2898, 2899, 2900, 2901 Extra Details: additive : EDTA(0.1 mM), point mutations; glycine; Escherichia coli dihydrofolate reductase;,stability; free energy; flexible loop

Submission Details

ID: oThaLjTP

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Gekko K;Yamagami K;Kunori Y;Ichihara S;Kodama M;Iwakura M,J. Biochem. (1993) Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase. PMID:8454578
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
6CW7 2018-03-30T00:00:00+0000 1.03 E. coli DHFR product complex with (6S)-5,6,7,8-TETRAHYDROFOLATE
6CXK 2018-04-03T00:00:00+0000 1.11 E. coli DHFR substrate complex with Dihydrofolate
6CYV 2018-04-06T00:00:00+0000 1.3 E. coli DHFR ternary complex with NADP and dihydrofolate
1DDR 1995-06-29T00:00:00+0000 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
1DDS 1995-06-29T00:00:00+0000 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
1DHI 1993-10-29T00:00:00+0000 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DHJ 1993-10-29T00:00:00+0000 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DRA 1991-11-06T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1DRB 1991-11-06T00:00:00+0000 1.96 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1DRE 1996-11-28T00:00:00+0000 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
96.2 Dihydrofolate reductase P31073 DYR_CITFR
91.8 Dihydrofolate reductase P31074 DYR_KLEAE