Exploring substrate specificity in the UbiD heteroaromatic decarboxylase PA0254

  • Herkus Miscikas

Student thesis: Master of Philosophy


The UbiD enzyme family of (de)carboxylases is dependent on a prenylated flavin mononucleotide (prFMN) cofactor. This cofactor is able to support an unusual 1,3 dipolar cycloaddition mechanism underpinning reversible decarboxylation. The widespread UbiD enzymes act on a range of unsaturated acids, but what governs substrate specificity within this family is not well understood. Here we explore the UbiD decarboxylase PA0254, which is capable of decarboxylating pyrrole-2-carboxylate. Previous studies obtained a crystal structure of the wild-type PA0254, with insights into the active site structure used to create a variant that can accept furan-2-carboxylate. We set out to create additional variants that would be able to accommodate a wide range of substrates, ideally including those with a high degree of aromaticity. Variants created were screened for activity with 4 heteroaromatic substrates – pyrrole-2-decarboxylate, furan-2-decarboxylate, thiophene-2-decarboxylate and indole-3-decarboxylate. Unfortunately, variants made yielded little to no activity. This suggests the altered residues could be implicated in substrate binding, cofactor binding/maturation or catalysis. To rationalise the activity of the N318C variant with furan-2-carboxylate, we set out to determine a crystal structure of this variant. This proved a non-trivial task due to crystal twinning problems encountered. The N318C crystal structure revealed no significant structural changes are introduced by the mutation. Our studies suggest rational engineering of UbiD enzymes to expand substrate scope is limited by multiple roles of active site residues, limiting the approach to modest expansions of substrate repertoire.
Date of Award31 Dec 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndrew Munro (Supervisor) & David Leys (Supervisor)


  • bioplastics
  • crystallography
  • UbiD
  • UbiX
  • prFMN

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