Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

Area of Science:

• Enzymology
• Structural Biology
• Antimicrobial Resistance

Background:

• Type II R67 dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme conferring trimethoprim resistance.
• R67 DHFR possesses a unique tetrameric structure with a single active site tunnel, distinct from chromosomal DHFR.
• Its architecture presents an atypical model for studying enzyme evolution and active site residue function.

Purpose of the Study:

• To investigate the functional contribution of individual protomers within the R67 DHFR tetramer.
• To explore the enzyme's tolerance for active-site substitutions and their impact on catalysis and stability.
• To determine the minimum requirement for native-like catalytic activity.

Main Methods:

• Library selection was employed to identify R67 DHFR variants with mutated active sites.
• Catalytic activity (kcat, KM) and thermal stability (Tm) of selected variants were assessed.
• Comparison of kinetic and stability parameters between native and mutated tetramers.

Main Results:

• Library selection favored variants maintaining two native protomers ('half-native' tetramers).
• These 'half-native' variants exhibited native-like KM but 5- to 33-fold lower kcat values.
• Mutated variants showed reduced thermal stability but increased resilience to heat denaturation.

Conclusions:

• Two native protomers are sufficient to confer near-native catalytic rates in R67 DHFR.
• The enzyme demonstrates significant tolerance for active-site substitutions.
• These findings provide insights into enzyme evolution and the structural basis of trimethoprim resistance.