A bacterial selection for the directed evolution of pyruvate aldolases.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Griffiths, JS; Cheriyan, M; Corbell, JB; Pocivavsek, L; Fierke, CA; Toone, EJ

Published Date

  • August 2004

Published In

Volume / Issue

  • 12 / 15

Start / End Page

  • 4067 - 4074

PubMed ID

  • 15246084

Pubmed Central ID

  • 15246084

Electronic International Standard Serial Number (EISSN)

  • 1464-3391

International Standard Serial Number (ISSN)

  • 0968-0896

Digital Object Identifier (DOI)

  • 10.1016/j.bmc.2004.05.034

Language

  • eng