title: Engineering strictosidine synthase: rational design of a small, focused circular permutation library of the β-propeller fold enzyme.
abstract: Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed β-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.
authors.affiliation: Department of Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Fischereder
authors.affiliation: Department of Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria; ACIB GmBH, c/o, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.
authors.forename: Desiree
authors.initials: D
authors.lastname: Pressnitz
authors.affiliation: Department of Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria. Electronic address: wolfgang.kroutil@uni-graz.at.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kroutil
authors.affiliation: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA. Electronic address: sal2@emory.edu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lutz
mesh_terms: D019759:Carbon-Nitrogen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D008958:Models, Molecular; D015394:Molecular Structure; D019151:Peptide Library; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011926:Rauwolfia / Q000201:enzymology*
chemical_list: D019151:Peptide Library; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase
keywords: Beta propeller protein; Biocatalysis; Bioorganic chemistry; Circular permutation; Protein engineering