title: Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97-27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay.
abstract: Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97-27 (hereafter, B. thuringiensis 97-27). The wtAsbF is relatively unstable at 37 °C, in vitro (t1/237= 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t1/237= 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 105M-1s-1, 7.8 × 105M-1s-1, respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, in vivo overexpression of Mut1 in E. coli resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80-120% increase in DHB accumulation in the media relative to the wild-type enzyme.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , P.O. Box 1663, MS M888, Los Alamos, New Mexico 87545, United States.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Harrington
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , P.O. Box 1663, MS M888, Los Alamos, New Mexico 87545, United States.
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , P.O. Box 1663, MS M888, Los Alamos, New Mexico 87545, United States.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Kern
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , P.O. Box 1663, MS M888, Los Alamos, New Mexico 87545, United States.
authors.forename: Emily N
authors.initials: EN
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, Northern Arizona University , P.O. Box 5698, Flagstaff, Arizona 86001, United States.
authors.forename: Gustavo M
authors.initials: GM
authors.lastname: Canales
authors.affiliation: Department of Chemistry, Northern Arizona University , P.O. Box 5698, Flagstaff, Arizona 86001, United States.
authors.forename: Kellan B
authors.initials: KB
authors.lastname: Finney
authors.affiliation: Department of Chemistry, Northern Arizona University , P.O. Box 5698, Flagstaff, Arizona 86001, United States.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Koppisch
authors.affiliation: Bioscience Division, Los Alamos National Laboratory , P.O. Box 1663, MS M888, Los Alamos, New Mexico 87545, United States.
authors.forename: Charlie E M
authors.initials: CE
authors.lastname: Strauss
authors.affiliation: Chemistry Division, Los Alamos National Laboratory , P.O. Box 1663, MS E554, Los Alamos, New Mexico 87545, United States.
authors.forename: David T
authors.initials: DT
authors.lastname: Fox
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D015698:Genomic Library; D057166:High-Throughput Screening Assays; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D065288:Serogroup; D058615:Synthetic Biology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; C044921:3-dehydroshikimate dehydratase; D006836:Hydro-Lyases
keywords: commodity chemicals; enzyme engineering; flow cytometry; shikimate pathway; thermostabilization