title: Differential effects of a mutation on the normal and promiscuous activities of orthologs: implications for natural and directed evolution.
abstract: Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder.
authors.forename: Akhil
authors.initials: A
authors.lastname: Khanal
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder.
authors.forename: Sean
authors.initials: S
authors.lastname: Yu McLoughlin
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder.
authors.forename: Jamie P
authors.initials: JP
authors.lastname: Kershner
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder shelley.copley@colorado.edu.
authors.forename: Shelley D
authors.initials: SD
authors.lastname: Copley
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D040961:Genetic Drift; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny
chemical_list: D001426:Bacterial Proteins; D005973:Glutamine; C061433:gamma-glutamyl phosphate; D010088:Oxidoreductases
keywords: ProA; enzyme evolution; epistasis; molecular evolution