title: Structure-based directed evolution of a monomeric triosephosphate isomerase: toward a pentose sugar isomerase.
abstract: Through structure-based and directed evolution approaches, a new catalytic activity has been established on the (β/α)8 barrel enzyme triosephosphate isomerase (TIM). This work started from ml8bTIM, a monomeric variant of TIM, in which the phosphate-binding loop (loop-8) had been shortened. Structure analysis suggested an additional point mutation (V233A), converting ml8bTIM into A-TIM. A-TIM has no detectable TIM activity, but it binds the TIM transition state analog, 2-phosphoglycollate. In an in vivo selection approach, we aimed at transferring the activity of three sugar isomerases (L-arabinose isomerase (L-AI), D-xylose isomerase A (D-XI) and D-ribose-5-phosphate isomerase (D-RPI)) onto A-TIM. Escherichia coli knockout variants were constructed, lacking E. coli L-AI, D-XI and D-RPI activities, respectively. Through a systematic approach, new A-TIM variants were obtained only from selection experiments with the L-AI knockout strain. Selection for D-RPI activity was impossible because of an impaired strain due to the gene knockouts. The selection for D-XI activity was unsuccessful, showing the importance of the starting protein for obtaining new biocatalytic properties. The L-AI-directed evolution experiments show that A-TIM already has residual in vivo L-AI activity. Most of the mutations providing A-TIM with enhanced L-AI activity are located in the loops between β-strands and the subsequent α-helices.
authors.affiliation: Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Insitute of Biotechnology, Ackerstr. 76, ACK 24, D-13355 Berlin, Germany mirja.krause@tu-berlin.de mirja.krause@oulu.fi.
authors.forename: Mirja
authors.initials: M
authors.lastname: Krause
authors.affiliation: Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Insitute of Biotechnology, Ackerstr. 76, ACK 24, D-13355 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Rik K
authors.initials: RK
authors.lastname: Wierenga
mesh_terms: D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D008958:Models, Molecular; D010429:Pentoses / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010429:Pentoses; D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase
keywords: directed evolution; protein engineering; structure-based rational design; sugar isomerases; triosephosphate isomerase