Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.
Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, H3C 3J7, Canada; PROTEO, the Québec Network for Protein Function, Structure and Engineering, Québec, Canada; CGCC, the Center for Green Chemistry and Catalysis, Montréal, Canada.
Maximilian C C J C
MC
Ebert
Krista L
KL
Morley
Jordan P
JP
Volpato
Andreea R
AR
Schmitzer
Joelle N
JN
Pelletier
D020134:Catalytic Domain / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004795:Enzyme Stability / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011489:Protein Denaturation; D055503:Protein Multimerization; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim
D014295:Trimethoprim; C098921:dihydrofolate reductase type II; D013762:Tetrahydrofolate Dehydrogenase
active site engineering; combinatorial mutations; homotetramer; thermostability; trimethoprim resistance