Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase.

Enviado por Abel Baerga-Ortiz el

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TítuloDirected mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase.
Publication TypeJournal Article
Year of Publication2006
AutoresBaerga-Ortiz, A, Popovic, B, Siskos, AP, O'Hare, HM, Spiteller, D, Williams, MG, Campillo, N, Spencer, JB, Leadlay, PF
JournalChem Biol
Volume13
Issue3
Pagination277-85
Date Published2006 Mar
ISSN1074-5521
Palabras claveAmino Acid Sequence, Catalysis, Molecular Conformation, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxidoreductases, Polyketide Synthases, Protein Structure, Tertiary, Recombinant Proteins, Stereoisomerism, Substrate Specificity, Time Factors
Abstract

The ketoreductase (KR) domains eryKR(1) and eryKR(2) from the erythromycin-producing polyketide synthase (PKS) reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Modeling of eryKR(1) and eryKR(2) showed that conserved amino acids previously correlated with production of alternative alcohol configurations lie in the active site. eryKR(1) domains mutated at these positions showed an altered stereochemical outcome in reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester. The wild-type eryKR(1) domain exclusively gave the (2S, 3R)-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester, while the double mutant (F141W, P144G) gave only the (2S, 3S) isomer, a switch of the alcohol stereochemistry. Mutation of the eryKR(2) domain, in contrast, greatly increased the proportion of the wild-type (2R, 3S)-alcohol product. These data confirm the role of key residues in stereocontrol and suggest an additional way to make rational alterations in polyketide antibiotic structure.
DOI10.1016/j.chembiol.2006.01.004
Alternate JournalChem. Biol.
PubMed ID16638533