کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
69481 | 48771 | 2015 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha](/preview/png/69481.png)
• The thiolase ReBktB shows activity on N-acetylcysteamine bound substrates.
• ReBktB shows a preference for the five-carbon β-ketoacyl substrate.
• The 2.0-Å resolution crystal structure of ReBktB was solved with 16 monomers/ASU.
• The structure helped to analyze handle and acyl group promiscuity.
• A mutant active towards α-methyl substituted substrates was rationally engineered.
Thiolases catalyze the formation of carbon–carbon bonds in diverse biosynthetic pathways. The promiscuous β-ketoacyl thiolase B of Ralstonia eutropha (ReBktB) has been utilized in the in vivo conversion 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 biocatalysts in vitro if 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 four ReBktB 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 of ReBktB and its engineered mutants to utilize NAC-linked substrates will facilitate the in vitro biocatalytic synthesis of diketide chiral building blocks from feedstock molecules such as acetate and propionate.
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Journal: Journal of Molecular Catalysis B: Enzymatic - Volume 121, November 2015, Pages 113–121