A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant

Abstract

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV. We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis. In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.

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

“…As we have previously reported, heterologous expression of the class Id NrdB protein from F. ignava (subclass NrdBi [13]) in the presence of excess Mn 2+ ions generates a form of the protein featuring a multiline EPR signal attributable to a Mn 2 (IV,III) cofactor [12]. To further probe the metal cofactor in this protein and elucidate its assembly mechanism we isolated a series of different forms of the F. ignava NrdBi protein.…”

“…In the case of class Ie, the metal cofactor is replaced with a single 3,4-dihydroxyphenylalanine (DOPA) organic radical [8, 9]. Conversely, class Id RNRs from Facklamia ignava , Leeuwenhoekiella blandensis and Flavobacterium johnsoniae are all proposed to function without an organic radical, and instead utilize a mixed valent Mn 2 (IV,III) metal centre [10–12]. However, this remains to be firmly established.…”

“…Still, their NrdB domain structures differ. The F. ignava NrdB has a unique domain structure comprising an N-terminal glutaredoxin (Grx) domain, followed by an ATP-cone domain and a radical-generating domain [12], whereas the L. blandensis NrdB consists of an N-terminal ATP-cone domain followed by the radical-generating domain [11] and the F. johnsoniae NrdB has no additional domains [10].…”

Reannotation of the Ribonucleotide Reductase in a Cyanophage Reveals Life History Strategies Within the Virioplankton

“…For Class I α-only and Class I β-only phylogenies, sequences were trimmed near the N-terminus to exclude evolutionarily mobile ATP cone domains (Aravind et al, 2000). Class I β sequences were also trimmed near the C-terminus to exclude any fused glutaredoxin domains (Rozman Grinberg et al, 2018b). In all cases, trimming was guided by annotated Class I subclass (a-e) and Class II subtype (mono- or dimeric) representatives (Supplementary Table 1).…”

Reannotation of the ribonucleotide reductase in a cyanophage reveals life history strategies within the virioplankton

“…For Class I α-only and Class I β-only phylogenies, sequences were 208 trimmed near the N-terminus to exclude evolutionarily mobile ATP cone domains (Aravind et al, 209 2000). Class I β sequences were also trimmed near the C-terminus to exclude any fused glutaredoxin 210 domains (Rozman Grinberg et al, 2018b). In all cases, trimming was guided by annotated Class I 211 subclass (a-e) and Class II subtype (mono-or dimeric) representatives (Table 2).…”