Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function.


Abstract

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His). The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V. The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme. The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction. Study holds ProTherm entries: 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919 Extra Details: additive : EDTA(0.1 mM), Activity at 25 degree C point mutations; glycine; Escherichia coli dihydrofolate reductase;,stability; free energy; flexible loop; function

Submission Details

ID: aTfUsK3U4

Submitter: Connie Wang

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

Version: 1

Publication Details
Gekko K;Kunori Y;Takeuchi H;Ichihara S;Kodama M,J. Biochem. (1994) Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function. PMID:7798183
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
91.8 Dihydrofolate reductase P31074 DYR_KLEAE
96.2 Dihydrofolate reductase P31073 DYR_CITFR
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6