Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression.
Three different forms of dihydrofolate reductase (DHFR) from Escherichia coli with amino acid replacements Thr35----Asp, Asn37----Ser and Arg57----His, and one form containing all three of these changes were obtained by oligonucleotide-directed mutagenesis. These amino acids are on the surface of the protein and two of them (Thr35 and Arg57) are invariant for known sequences of DHFR. Conversion of Asn37----Ser has no effect on the functional activity or the protein level in the cells. The Thr35----Asp replacement leads to a sharp decrease in the protein level, while the addition of a DHFR inhibitor, trimethoprim (Tmp), to the growth medium increases the level of DHFR in the cells. There is a very small quantity of DHFR with all three amino acid changes. The addition of Tmp to the growth medium also leads to an increase in the mutant protein levels. The mutant with the Arg57----His replacement renders the cells sensitive to Tmp, but the level of DHFR is the same as for the wild-type protein. It is suggested that the invariant Thr35 is important for the stable conformation of DHFR whereas Arg57 is essential for protein activity. Various structural and functional aspects of these results are discussed.
Murzina
N V
NV
Institute of Protein Research, Academy of Sciences, Moscow Region, USSR.
Gudkov
A T
AT
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005493:Folic Acid Antagonists; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology
D005493:Folic Acid Antagonists; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase