期刊名称：Computational and Structural Biotechnology Journal
印刷版ISSN：2001-0370
出版年度：2021
卷号：19
页码：3087-3096
DOI：10.1016/j.csbj.2021.05.031
出版社：Computational and Structural Biotechnology Journal
摘要：The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C 8 ) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p -nitrophenyl alkanoate esters as surrogate substrates. In the present study, p -nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p -Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p -nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p -nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p -nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p -nitrophenyl 1- 13 C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p -nitrophenyl esters.
