Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations.
We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.
Schmitzer
Andreea R
AR
Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, H3C 3J7, Montréal, Québec, Canada.
Lépine
François
F
Pelletier
Joelle N
JN
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D020650:Combinatorial Chemistry Techniques; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D000596:Amino Acids; D019151:Peptide Library; D013762:Tetrahydrofolate Dehydrogenase