Mechanism of a Standalone β-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme

Serina L. Robinson, James K Christenson, Jack E Richman, Dominick J. Jenkins, João Neres, Dallas R. Fonseca, Courtney Aldrich, Lawrence P Wackett

Research output: Contribution to journalArticle

Abstract

Enzyme-catalyzed β-lactone formation from β-hydroxy acids is a crucial step in bacterial biosynthesis of β-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β-lactone synthetase activity using synthetic β-hydroxy acid substrates with alkene or alkyne moieties. β-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain β-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.

Original languageEnglish (US)
Pages (from-to)1701-1711
Number of pages11
JournalChemBioChem
Volume20
Issue number13
DOIs
StatePublished - Jul 1 2019

Fingerprint

Lactones
Ligases
Assays
Enzymes
Xanthomonas campestris
Substrates
Hydroxy Acids
Coenzyme A Ligases
Mutagenesis
Hydroxylamine
Decarboxylation
Stereoisomerism
Alkynes
Biosynthesis
Alkenes
Pathogens
Adenosine Monophosphate
Chromophores
Hydrocarbons
Substrate Specificity

Keywords

  • ANL superfamily
  • adenylation
  • beta-lactone synthetase
  • biosynthesis
  • natural products

PubMed: MeSH publication types

  • Journal Article

Cite this

Mechanism of a Standalone β-Lactone Synthetase : New Continuous Assay for a Widespread ANL Superfamily Enzyme. / Robinson, Serina L.; Christenson, James K; Richman, Jack E; Jenkins, Dominick J.; Neres, João; Fonseca, Dallas R.; Aldrich, Courtney; Wackett, Lawrence P.

In: ChemBioChem, Vol. 20, No. 13, 01.07.2019, p. 1701-1711.

Research output: Contribution to journalArticle

Robinson, Serina L. ; Christenson, James K ; Richman, Jack E ; Jenkins, Dominick J. ; Neres, João ; Fonseca, Dallas R. ; Aldrich, Courtney ; Wackett, Lawrence P. / Mechanism of a Standalone β-Lactone Synthetase : New Continuous Assay for a Widespread ANL Superfamily Enzyme. In: ChemBioChem. 2019 ; Vol. 20, No. 13. pp. 1701-1711.
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