Thesis presented March 20, 2003
Abstract:
NAD(P)H:flavin oxidoreductase are ubiquitous enzyme. They catalyse the reduction of free flavins (riboflavin, FAD, FMN) by reduced pyridine nucleotides NAD(P)H as electron donor. Flavin reductase use flavin as substrate and not as cofactor. The real biological function of flavin reductase is still not well understood. Recently, a group of flavin reductase has been associated with hydroxylation reaction in combination with flavin dependent monooxygenase.
In a first part, we have worked on the Fre enzyme found in E. coli. The sequencing of E. coli genome reveals that Fre can play a role in ubiquinone biosynthesis in the hydroxylation of 2-octaprenylphenol. The experiments revealed that Fre is not implicated in ubiquinone biosynthesis, but that it could play a role in respiratory chain.
In a second part of this work, we have studied ActVB, a flavin reductase from Streptomyces coelicolor. ActVB is a NADH:flavin oxidoreducatse participating in the last step of actinorhodine biosynthesis. We have overexpressed and purified ActVB. The preparation of ActVB had highly varible amount of protein bound FMN. However, flavin reductase activities (FAD, FMN, riboflavin) do not depend on the amount of FMN bound to the protein. Act VB proceeds by an ordered sequential mechanism, where NADH is the first substrate and reduced flavin the last product. We have showed that both ribitol-phosphate chain and the isoalloxazine ring contribute to the protein flavin interaction.
ActVB, like other flavin reductase, can reduce the ferric iron in presence of NADH and free flavin. Here, we showed that ActVB can reduce ferric iron in the absence of free flavin because of the presence of FMN bound to the protein. The reduction of ferric iron by ActVB proceeds through a Ping-Pong mechanism.
We have identified the monooxygenase that is associated to ActVB in actinorhodine biosynthesis. ActVA-ORF5 was cloned and overexpressed in E. coli cell. We have established ActVA-ORF5 purification procedure. Finally, we found that the hydroxylation of 1,5 dihydroxyanthraquinone can occur only in presence of ActVB, FMN, NADH and oxygen.
Keywords:
ActVB, AD(P)H:flavin oxidoreductase, riboflavin, FAD, FMN, Fre enzyme, ubiquinone biosynthesis, actinorhodine biosynthesis, ActVA-ORF5, Streptomyces coelicolor