The in-biology novel high-valent manganese cofactor {MnIII MnIV} has been elucidated and characterized in a ribonucleotide reductase from Facklamia ignava, establishing the new radical-free ribonucleotide reductase class; class Id. The cofactor was found to be directly connected to catalytic activity and a mixed-metal cofactor was disproven using a newly formulated in vitro assembly protocol and activity assays. The cofactor was assembled in vitro using hydrogen peroxide, and it was shown that the F. ignava ribonucleotide reductase can perform catalase activity, with the cofactor formation as a side-reaction. Using electron paramagnetic resonance spectroscopy to study the in vitro assembled cofactor, a hydrogen peroxide-driven assembly mechanism was proposed, where the mechanism is like that of manganese catalases. An over-oxidized {MnIV MnIV} intermediate is suggested to be the direct precursor to the {MnIII MnIV} cofactor. The cofactor was also shown to be able to be assembled with superoxide, with in situ generation of superoxide using hydroquinone. An in vivo assembly mechanism was proposed to involve superoxide generated by some unknown accessory enzyme, similar to the NrdI assembly system of class Ib ribonucleotide reductases.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-500000 |
| Date | January 2018 |
| Creators | Berglund, Sigrid |
| Publisher | Uppsala universitet, Molekylär biomimetik |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | UPKEM E ; 168 |
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