PEDS Advance Access originally published online on December 2, 2004
Protein Engineering Design and Selection 2004 17(11):809-819; doi:10.1093/protein/gzh090
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Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations
1Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, H3C 3J7, Montréal and 2INRS–Institut Armand-Frappier, 531 Boulevard des Prairies, H7V 1B7, Laval, Québec, Canada
3 To whom correspondence should be addressed. E-mail: joelle.pelletier{at}umontreal.ca
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 kcat and KM values were similar to those for the native enzyme. The favourable 
(G) 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.
Received September 16, 2004; revised November 18, 2004; accepted November 19, 2004.
Edited by Jacques Fastrez
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