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

Megan D. Smith, Lambros J. Tassoulas, Troy A. Biernath, Jack E. Richman, Kelly G. Aukema, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)3087-3096
Number of pages10
JournalComputational and Structural Biotechnology Journal
Volume19
DOIs
StatePublished - Jan 2021

Bibliographical note

Funding Information:
This work was financially supported by National Institutes of Health Biotechnology training grant (5T32GM008347-27) and the University of Minnesota MnDRIVE program. We thank the U.S. Department of Energy Joint Genome Institute for synthetic DNA. The work conducted by the U.S. Department of Energy (DOE) Joint Genome Institute, a DOE Office of Science User Facility, is supported under [DE-AC02-05CH11231].

Funding Information:
This work was financially supported by National Institutes of Health Biotechnology training grant (5T32GM008347-27) and the University of Minnesota MnDRIVE program. We thank the U.S. Department of Energy Joint Genome Institute for synthetic DNA. The work conducted by the U.S. Department of Energy (DOE) Joint Genome Institute, a DOE Office of Science User Facility, is supported under [DE-AC02-05CH11231]. MDS and LPW conceived the study. MDS, LJT, TB, JER, LPW participated in research design. MDS, LJT, TB, JER performed the experiments. All authors analyzed data. MDS and LPW wrote the original draft of the manuscript and all authors approved the final version.

Publisher Copyright:
© 2021 The Authors

Keywords

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

PubMed: MeSH publication types

  • Journal Article

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