An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs

Abstract

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(-1)at 20 degrees C; DeltaT(m) = 3.0 degrees 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 degrees 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.

A new class of adenylate kinase in methanogens is related to uridylate kinase

“…The observed Km values were consistent with other AMP kinases, typically in the range of 2-100 lM for ATP and 11-600 lM for AMP (Lacher and Schafer 1993;Shamoo 2009, 2010;Scheer et al 2011). The observed specific activity for AdkB was lower than those reported for both the archaeal AdkA and the bacterial enzymes (14-1,400 lmol min -1 mg -1 ) (Rusnak et al 1995;Counago et al 2008;Shamoo 2009, 2010). As noted, the kinetic parameters reported above were determined utilizing the coupled assay at 37°C; however, as predicted by the thermophilic nature of M. jannaschii, the specific activity of AdkB greatly increases with temperature.…”

Performance of protein stability predictors

“…The active-site residues of enzymes are generally polar or charged, and are usually located in hydrophobic clefts [Fersht, 1999]. Stabilizing mutations in active site residues can reduce enzymatic activities [Beadle and Shoichet, 2002;Counago et al, 2008;Garcia et al, 2000;Kidokoro et al, 1995;Meiering et al, 1992;Mukaiyama et al, 2006;Nagatani et al, 2007;Schreiber et al, 1994, Shoichet et al, 1995Zhi et al, 1991]. Additionally, a stabilizing mutation increased the resistance of ribonuclease A to proteolysis [Markert et al, 2001], which, for example, would be an undesirable effect if it occurred in enzymes involved in cell signaling [Fink, 2005].…”

Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli

“…Currently, study of adaptive constraints at the molecular level has mostly been limited to directed protein evolution studies that have examined how mutations selected to alter specific enzyme properties achieve that effect (49,(53)(54)(55)(56)(57)(58)(59). This is one of a handful of studies to date to examine the molecular basis underlying the effect of a set of adaptive mutations to the same gene on organism fitness (2,60,61).…”