A bacterial selection for the directed evolution of pyruvate aldolases

Bioorganic & Medicinal Chemistry
Jennifer S GriffithsEric J Toone

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.

Citations

Apr 15, 2006·Applied Microbiology and Biotechnology·Anne K Samland, Georg A Sprenger
Jun 3, 2006·Natural Product Reports·Andrew S Rowan, Chris J Hamilton
Oct 3, 2006·Applied and Environmental Microbiology·Helge HenningKarl-Erich Jaeger
Feb 1, 2006·Proceedings of the National Academy of Sciences of the United States of America·Stephan van Sint FietBernard Witholt
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Apr 3, 2008·Chembiochem : a European Journal of Chemical Biology·Ykelien L BoersmaWim J Quax
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Jan 31, 2008·Archives of Biochemistry and Biophysics·Amanda BoltAdam Nelson
Sep 29, 2011·Bioorganic & Medicinal Chemistry·Manoj CheriyanCarol A Fierke

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