title: Directed evolution of the substrate specificity of biotin ligase.
abstract: We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications.
authors.affiliation: Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas.
authors.forename: Wei-Cheng
authors.initials: WC
authors.lastname: Lu
authors.forename: Matthew
authors.initials: M
authors.lastname: Levy
authors.forename: Rodney
authors.initials: R
authors.lastname: Kincaid
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D019943:Amino Acid Substitution; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D020125:Mutation, Missense; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D001710:Biotin; C004749:desthiobiotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: biotin; biotin protein ligase; desthiobiotin; emulsion; in vitro compartmentalization