title: A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.
abstract: The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.
authors.affiliation: Institute of Analytical Chemistry, Chemo- and Biosensors and ‡Institute of Biophysics and Physical Biochemistry, University of Regensburg , 93040 Regensburg, Germany.
authors.forename: Raphaela B
authors.initials: RB
authors.lastname: Liebherr
authors.forename: Max
authors.initials: M
authors.lastname: Renner
authors.forename: Hans H
authors.initials: HH
authors.lastname: Gorris
mesh_terms: D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D005966:Glucuronidase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D005966:Glucuronidase