title: How do mutations at phenylalanine-153 and isoleucine-155 partially suppress the effects of the aspartate-27-->serine mutation in Escherichia coli dihydrofolate reductase?
abstract: Several second-site suppressors of the D27S lesion in Escherichia coli dihydrofolate reductase (DHFR) have been identified. The activity of the primary mutant, D27S DHRF, was found to be greatly decreased at pH 7.0, consistent with aspartic acid-27 being critically involved in proton donation during catalysis. Partial suppressors of the D27S mutation have been selected by their ability to confer an increased resistance to trimethoprim upon host E. coli; the suppressors have been identified as F153S or I155N substitutions. D27S+F153S and D27S+I155N DHFRs display 2-3-fold increases in kcat over D27S DHFR values, but only the F153S mutation decreases the Km for dihydrofolate by a factor of 2. Neither double mutant approaches wild-type DHFR activity. Unexpectedly, Phe153 and Ile155 occur on the surface of the protein and are approximately 8 and 14 A distant from the active site. Ile155 is a member of a beta-bulge. A previously identified suppressing mutation, F137S, occurs nearby and is also a member of the same beta-bulge [Howell et al. (1990) Biochemistry 29, 8561-8569]. Clustering of these three second-site mutations indicates this area of the structure may be important in protein function. Conformational changes due to the presence of these suppressing mutations are likely as the F153S and I155N mutations do not affect hydride-transfer rates upon introduction in wild-type DHFR and alterations in circular dichroism spectra are associated with the double-mutant DHFRs.
authors: Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
authors: A
authors: A
authors: Dion
authors: C E
authors: CE
authors: Linn
authors: T D
authors: TD
authors: Bradrick
authors: S
authors: S
authors: Georghiou
authors: E E
authors: EE
authors: Howell
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D007532:Isoleucine; D010649:Phenylalanine; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase