Exploring aromatic acid (de)carboxylases: The UbiD family

UoM administered thesis: Phd

  • Authors:
  • Stephen Marshall

Abstract

Global warming and depletion of fossil fuels dictate that we search for alternative and carbon neutral sources of fuel and chemicals. As part of a wider toolbox of catalysts, reversible decarboxylases are potentially important in the conversion of renewable feedstocks to the desired end products. Their application however requires further fundamental understanding of mechanism and substrate scope. Members of the UbiD family of decarboxylases utilise a prenylated FMN cofactor produced by the partner protein UbiX from FMNH2 and the non-archetypal isoprenoid precursor dimethylallyl monophosphate (DMAP). The prFMN is essential for the transient cycloaddition reaction that underpins (de)carboxylation of aliphatic alkene substrates. It is unclear whether a similar mechanism supports (de)carboxylation reactions on aromatic substrates (given the inherent stability of the aromatic ring), and this forms the subject of this thesis. Furthermore, additional subunits have been identified as part of the UbiD/X system, and we here seek to explore their role in catalysis. Our studies on two aromatic acid (de)carboxylases, the canonical UbiD and AroY, reveal unexpected variation occurs in terms of cofactor maturation. The UbiD enzyme was reconstituted in vitro with reduced prFMN, but oxidative maturations stalls at a prFMNsemiquinone radical species. In contrast, successful oxidative maturation of AroY, a protocatechuic acid decarboxylase, occurred in vitro. Crystal structures of both enzymes suggest the possibility of domain motion(s) linked to prFMN and substrate binding. Unfortunately, ligand complex structures could not be obtained, limiting our mechanistic understanding at present. Given the difficulties in obtaining active UbiD, it is possible that cofactor binding and maturation requires additional factors in vivo. We reveal that one of the smaller UbiD/X associated proteins, LpdD, is a prFMN binding protein. Structural similarities between LpdD and a yeast chaperone protein suggest LpdD assists with transfer of reduced prFMN from UbiX to UbiD. This data provides further insights into the UbiD enzymes, contributing to a complete understanding of the scope for these proteins to be used in future biocatalysis.

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Original languageEnglish
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Award date31 Dec 2017