PEDS Advance Access published online on December 19, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm072
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An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability–activity trade-offs
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, MS-140 Houston, TX, USA
3 To whom correspondence should be addressed. E-mail: shamoo{at}rice.edu
Protein function is a balance between activity and stability. However, the relevance of stability–activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine. Previously, we have linked organismal survival at increasing temperatures to adaptive changes to a single protein sequence through allelic replacement of an essential gene, adenylate kinase (adk), in a thermophile. In vivo continuous evolution of the temperature-sensitive thermophile has shown that the first step toward increased organismal fitness is mutation of glutamine-199 to arginine in the mesophilic enzyme (AKsub Q199R). Here, we show that although substitution of Arg-199 did confer a modest increase in stability (0.6 kcal mol–1at 20°C; 
Tm = 3.0°C), it is a large change in the activity profile of the enzyme that is responsible for its exceptional robustness during the earlier experimental evolution study. Kinetic studies of AKsub Q199R show that it has a strong loss of enzymatic activity (>50%) at lower temperatures (20–45°C) and a subsequent increase at elevated temperatures. The stability–activity trade-off observed for AKsub Q199R was linked to the rigidification of the overall structure through stabilization of a polypeptide loop containing Arg-199 that is part of the ATP-binding site of the enzyme. Structural analysis revealed the formation of new ionic interactions facilitated by Arg-199. Our results suggest that stability–activity trade-offs are employed readily as an evolutionary strategy during natural selection to increase organismal fitness.
Keywords: adaptive evolution/adenylate kinase/molecular evolution/thermostability/trade-offs
Received May 21, 2007; revised November 9, 2007; accepted November 12, 2007.
1 Present address: Department of Biochemistry, University of Otago, New Zealand
2 Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA