title: A bacterial selection for the directed evolution of pyruvate aldolases.
abstract: A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.
authors.affiliation: Department of Chemistry, Duke University, Box 90317, Durham, NC 27708, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Griffiths
authors.forename: Manoj
authors.initials: M
authors.lastname: Cheriyan
authors.forename: Jayme B
authors.initials: JB
authors.lastname: Corbell
authors.forename: Luka
authors.initials: L
authors.lastname: Pocivavsek
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Toone
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011773:Pyruvates; D011770:Pyruvate Kinase; D000446:Aldehyde-Lyases