Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

Inna Rozman Grinberg,Mahmudul Hasan,Gustav Berggren,Derek T Logan,Sigrid Berglund,Felix M Ho,Daniel Lundin,Britt-Marie Sjöberg,Ann Magnuson

doi:10.1007/s00775-019-01697-8

Inna Rozman Grinberg, Mahmudul Hasan + Show 7 more

Open Access

https://doi.org/10.1007/s00775-019-01697-8

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Abstract

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.

Highlights

Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotides
As we have previously reported, heterologous expression of the class Id NrdB protein from F. ignava in the presence of excess M n2+ ions generates a form of the protein featuring a multiline EPR signal attributable to a Mn2(IV,III) cofactor [12]
The data presented strongly support the dependence on a Mn2(IV,III) cofactor for efficient catalysis of F. ignava class I RNR

Summary

Introduction

Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotides (dNTPs). As such, it is an essential enzyme found in all species apart from a few intracellular parasites, and it is even encoded in certain dsDNA viruses to ensure ready access to DNA building blocks. RNRs form an enzyme family currently comprising three different classes and several subclasses. All RNRs share a common reaction mechanism, in which a cysteine radical induces the reduction of ribonucleotides, but they differ in the way the radical mechanism is initiated [1,2,3,4,5]. Class I RNRs consist of a substrate-binding component (NrdA or α) and a radical-generating component (NrdB or β). The β-subunit is dimeric (β2) and the active complex is a heterotetramer (α2β2) in bacteria and a heterooctamer (α6β2) in eukaryotes

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