p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA

https://doi.org/10.1016/j.csbj.2021.05.031Get rights and content
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Abstract

The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) 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 1-13C-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.

Graphical abstract

The thiolase enzyme OleA initiates the biosynthesis of β-lactone natural products and membrane hydrocarbons via acyl-Coenzyme A (CoA) substrates, but this study showed that p-nitrophenyl alkanoates can substitute, yielding new insights into the enzyme mechanism and providing a cheaper and more available starting substrate.

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Keywords

OleA
Thiolase
Claisen condensation
Bacteria
p-Nitrophenyl ester
Acyl-CoA
β-Lactone
Membrane hydrocarbon
Natural product

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