Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase fromRalstonia eutropha.
Thiolases catalyze the formation of carbon-carbon bonds in diverse biosynthetic pathways. The promiscuous β-ketoacyl thiolase B ofRalstonia eutropha(ReBktB) has been utilized in thein vivoconversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into β-hydroxy acids, including the pharmaceutically-important 3,4-dihydroxybutyric acid. Such thiolases could serve as powerful carbon-carbon bond-forming biocatalystsin vitroif handles less costly than CoA were employable. Here, thiolase activity is demonstrated toward substrates linked to the readily-available CoA mimic,N-acetylcysteamine (NAC).ReBktB was observed to catalyze the retro-Claisen condensation of several β-ketoacyl-S-NAC substrates, with a preference for 3-oxopentanoyl-S-NAC over 3-oxobutanoyl-, 3-oxohexanoyl-, and 3-oxoheptanoyl-S-NAC. A 2.0 Å-resolution crystal structure, in which the asymmetric unit consists of fourReBktB tetramers, provides insight into acyl group specificity and how it may be engineered. By replacing an active site methionine with an alanine, a mutant possessing significant activity towards α-methyl substituted, NAC-linked substrates was engineered. The ability ofReBktB and its engineered mutants to utilize NAC-linked substrates will facilitate thein vitrobiocatalytic synthesis of diketide chiral building blocks from feedstock molecules such as acetate and propionate.
Department of Molecular Biosciences, The University of Texas, Austin, TX 78712, USA.
Christopher D
CD
Fage
Department of Molecular Biosciences, The University of Texas, Austin, TX 78712, USA.
Jessica L
JL
Meinke
Department of Molecular Biosciences, The University of Texas, Austin, TX 78712, USA. ; Department of Chemistry, The University of Texas, Austin, TX 78712, USA.
Adrian T
AT
Keatinge-Clay