Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties.
Ancestral sequence reconstruction has been widely used to study historical enzyme evolution, both from biochemical and cellular perspectives. Two properties of reconstructed ancestral proteins/enzymes are commonly reported--high thermostability and high catalytic activity--compared with their contemporaries. Increased protein stability is associated with lower aggregation rates, higher soluble protein abundance and a greater capacity to evolve, and therefore, these proteins could be considered "superior" to their contemporary counterparts. In this study, we investigate the relationship between the favourable in vitro biochemical properties of reconstructed ancestral enzymes and the organismal fitness they confer in vivo. We have previously reconstructed several ancestors of the enzyme LeuB, which is essential for leucine biosynthesis. Our initial fitness experiments revealed that overexpression of ANC4, a reconstructed LeuB that exhibits high stability and activity, was only able to partially rescue the growth of a ΔleuB strain, and that a strain complemented with this enzyme was outcompeted by strains carrying one of its descendants. When we expanded our study to include five reconstructed LeuBs and one contemporary, we found that neither in vitro protein stability nor the catalytic rate was correlated with fitness. Instead, fitness showed a strong, negative correlation with estimated evolutionary age (based on phylogenetic relationships). Our findings suggest that, for reconstructed ancestral enzymes, superior in vitro properties do not translate into organismal fitness in vivo. The molecular basis of the relationship between fitness and the inferred age of ancestral LeuB enzymes is unknown, but may be related to the reconstruction process. We also hypothesise that the ancestral enzymes may be incompatible with the other, contemporary enzymes of the metabolic network.
School of Science, Faculty of Science and Engineering, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand. jhobbs@uvic.ca.|Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, STN CSC, Victoria, BC, V8W 3P6, Canada. jhobbs@uvic.ca.
Joanne K
JK
Hobbs
School of Science, Faculty of Science and Engineering, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
Erica J
EJ
Prentice
Laboratoire de Biometrie et Biologie Evolutive, Universite de Lyon, Universite Lyon 1, CNRS, UMR5558, Villeurbanne, France.|Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Mathieu
M
Groussin
School of Science, Faculty of Science and Engineering, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand. varcus@waikato.ac.nz.
Vickery L
VL
Arcus
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D019143:Evolution, Molecular; D056084:Genetic Fitness / Q000235:genetics*; D008958:Models, Molecular; D010802:Phylogeny; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins
3-Isopropylmalate dehydrogenase; Ancestral sequence reconstruction; Enzyme evolution; Fitness; Protein resurrection; Stability