Janus of the Flavin World : A Study of prFMN Cofactor Maturation and Catalysis

• Main Author: Balaikaite, Arune
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Amino acids; Analytical chemistry; Atomic physics; Biochemistry; Catalysis; Crystal structure; Dehydrogenases; Electrons; Hydrogen bonds; Mass spectrometry; Medical imaging; Mutation; NMR; Nuclear magnetic resonance; Oxidation; Scientific imaging; Vitamin B
• ISBN: 9798384195016

Flavins are an old family of cofactors, first discovered almost 150 years ago, in 1879. But even up to this day they do still bring surprises, revealing new structures and novel mechanisms. One such recent discovery is prenylated FMN (prFMN) – an old FMN cofactor dressed up by an additional 6-membered non-aromatic ring connecting N5 and C6 atoms. It is synthesized by a prenyltransferase UbiX, and then the cofactor is transferred to the UbiD-enzyme family members for the catalysis. Interestingly, UbiX releases a reduced prFMNH2 while UbiDs have been shown to use the oxidised prFMNiminium species. We have used the most well characterised UbiD - Aspergillus niger Fdc1 - as our model. Hence, Chapter 1 explores where / when the cofactor maturation occurs in Fdc1. Using UV/Vis and Raman spectroscopy, and mass spectrometry we demonstrate that prFMN maturation must occur within the Fdc1. Otherwise enzyme-free prFMNH2species are oxidised to the stable hydroperoxylated prFMN (prFMN C4a-OOH), that do not support the catalysis in Fdc1.Chapter 2 further explores the mechanism by which Fdc1 controls the maturation of prFMNH2. Arg173 is highly conserved residue and previously published literature demonstrated it being essential for the Fdc1 catalysis as it coordinates the substrate (α,β-unsaturated aliphatic acids). In contrast, Gln190 directly coordinates prFMN, and not the substrate. Hence, we have generated a range of Arg173 and Gln190 variants, and compared their cofactor maturation behaviour. Detailed UV/Vis spectrometry, mass spectrometry and crystallographic studies revealed that Arg173 most likely prevents prFMNiminiumhydroxylation, while Gln190 may ensure correct oxygen binding. None of the variants ware capable of both maturation and catalysis demonstrating the key role of the Arg173 and Gln190 in the cofactor maturation.N5-alkylated flavins have been predicted to have unique properties from the rest of the flavins, such as ability to stabilise (hydro)peroxy-group at C4a locus, as demonstrated in Chapter 1. This would produce a catalyst, akin to the flavin transitional state observed only in monooxygenases, but with more scope for engineering and exploring novel catalyses without the limitations of the original enzyme. In Chapter 3 we explore if prFMN is able to catalyse sulfide conversion to sulfoxide in presence of H2O2. Solution studies revealed that while the hydroperoxylated prFMN could not perform sulfoxidation, both FMN and prFMN demonstrated sulfide oxidation in presence of light. Addition of protein scaffold or another N5-alkylated flavin (N5- 9 Et RBF), as suggested by (de Gonzalo et al., 2011), did not increase the yield of sulfoxidation either.