PMC

Modern protein ribonucleotide reductases. A simplified scheme summarising the chemistry of ribonucleotide reduction for each of the three classes of ribonucleotide reductase. In the first step, a radical is generated at some distance from the active site. In class I and III, radical generation and catalysis occur on different subunits. The class I reductases produce a stable tyrosine radical with the help of an iron-oxygen centre, and this is transferred to the active site, 30–40 Å away. In classes II and III, a nucleotide cofactor (adenosylcobalamin (AdoCbl) and AdoMet, respectively) is cleaved to generate a deoxyadenosyl radical (AdoCH₂^•), which subsequently forms a stable glycine radical in the class III reductase. In the next step the radical in all three classes is transferred to an active site cysteine, forming a thiyl radical. This is then transferred to the 3′ position of the ribose, and two cysteines act as reductants in classes I and II, while formate is the reductant in the class III reaction. Once the 2′-OH has been reduced to 2′-H, with regeneration of the thiyl radical, this is transferred back to the tyrosine in class I, to the glycine in class III, while in class II AdoCbl is regenerated. In the class III reaction, AdoMet is consumed in generating the glycine radical, but like the tyrosine radical in the class I reaction, the glycine radical is stable and can presumably support several enzymatic turn-over cycles.

(Upper) Proteins of classes A–D contain a canonical radical SAM domain and two conserved Cys residues (class A), an N-terminus cobalamin binding domain (class B), a C-terminus HemN domain (class C) and a C-terminus methylenetetrahydrofolate domain (class D), respectively. (Lower) Proteins of class E contain a radical SAM domain flanked by conserved Cys and His residues that could potentially serve as FeS cluster-binding domains (left). This class of proteins (represented by NifB) do not contain a pair of conserved Cys residues of class A proteins (represented by RlmN and Cfr) based on partial sequence alignment (right). The blue arrow indicates the site of intermediary methylation in Rlm and Cfr (right). BD, binding domain.

All classes of RNRs catalyze the reduction of ribonucleotides to deoxyribonucleotides and require a metallo-cofactor to generate a thiyl radical that initiates this process. A, all RNRs have a structurally homologous α subunit (red) where nucleotide reduction occurs and is initiated by the thiyl radical. RNRs are classified based on the metallo-cofactors (blue) that oxidize the active site cysteine into the transient thiyl radical. This oxidation in the class I RNRs requires a second subunit, β (circled in black), designated NrdB for the Ia and NrdF for the Ib proteins. The oxidation uses AdoCbl for the class II RNR (NrdJ) and a glycyl radical for the class III RNR (NrdD). The glycyl radical is generated by an activating enzyme NrdG (black) that requires S-adenosylmethionine (SAM) and a [4Fe4S]⁺ cluster. In the class I and II RNRs, deoxynucleotide formation is accompanied by oxidation of two cysteines to a disulfide and consequently requires Trx, TrxR, and NADPH for multiple turnovers. NrdH-redoxin can function as a Trx in reduction of class Ib RNRs. In the class III RNRs, formate is the reductant. B, O₂ is required to assemble the active Mn^III₂-Y^• and Fe^III₂-Y^• cofactors of NrdF (class Ib RNR), which then act catalytically with NrdE to produce deoxynucleotides. The glycyl radical cofactor of the class III RNR (NrdD) is inactivated by O₂. C, structure of the AdoCbl cofactor used by the class II RNRs. Ado, adenosyl; AdoCbl, adenosylcobalamin; Cbi (in gray), cobinamide; DMB, dimethylbenzimidazole.

(A) Five classes of radical SAM methyltransferases (RSMTs) and (B) partial sequence alignment of members of class A and class E. Class A proteins have a canonical radical SAM domain and two conserved Cys residues that facilitate methyl transfer; class B proteins have a radical SAM domain and an N terminus cobalamin binding domain, and they can use methyl-cobalamin in methylation reactions; class C proteins have a radical SAM domain and a C terminus HemN domain that assists with substrate binding; and class D proteins have a radical SAM domain and a C terminus methylenetetrahydrofolate domain and, unlike proteins of classes A, B, and C, they may use methylenetetrahydrofolate instead of SAM as the source of the methyl carbon (, ). Class E proteins have a radical SAM domain and conserved Cys and His residues that serve as FeS cluster-binding domains, and they are presumably specialized in complex metallocluster assembly. BD, binding domain. Representative members of all classes are shown in A. The blue arrow in B indicates the site of intermediary methylation in Rlm and Cfr.