Effect of dimerization on the stability and catalytic activity of dihydrofolate reductase from the hyperthermophile Thermotoga maritima.
In contrast to all other chromosomally encoded dihydrofolate reductases characterized so far, dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima forms a highly stable dimer. The dimer interface involves residues whose mobility is important for catalysis in monomeric DHFRs. Here, we report the generation of a variant of DHFR from T. maritima, TmDHFR-V11D, in which a single amino acid replacement was sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric TmDHFR-V11D was 15 kJ mol(-1) lower than that of the wild-type dimer, while the melting temperature of monomeric TmDHFR-V11D was comparable to that of monomeric DHFR from the thermophile Bacillus stearothermophilus, supporting the hypothesis that oligomerization is required to achieve the thermal stabilities necessary for activity at temperatures optimal for growth of hyperthermophiles. Both the steady-state turnover numbers and rates of hydride transfer were reduced in TmDHFR-V11D. However, a similar reduction of the rate constants was observed in a different variant, TmDHFR-V126E, which remained as a dimer under all experimental conditions used here. Monomeric TmDHFR-V11D had a similar rate of hydride transfer to the dimeric form, but a reduced steady-state turnover rate. Intersubunit motions therefore appear to be less important than correlated motions within individual subunits for TmDHFR-catalyzed hydride transfer, but are critical to the overall progression of the catalytic cycle. Hence, the reduced catalytic activity of TmDHFR relative to the monomeric Escherichia coli enzyme is not caused by rigidity resulting from dimerization, but is a subtle consequence of the sequence and structure of its subunits, which appear to have evolved to allow thermostability at the expense of catalysis.
Loveridge
E Joel
EJ
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, United Kingdom.
Rodriguez
Robert J
RJ
Swanwick
Richard S
RS
Allemann
Rudolf K
RK
D019943:Amino Acid Substitution / Q000235:genetics; D000838:Anions; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D014644:Genetic Variation; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics*
D000838:Anions; D013762:Tetrahydrofolate Dehydrogenase