Radical Enzymes

Abstract

Radical enzymes harbour a free radical in their polypeptide chain, which participates in catalysis. A growing number of enzymes are known to require a posttranslationally generated free radical for their proper functioning. The radical is generally used to remove a hydrogen atom from an unreactive position in the substrate, activating the substrate to undergo difficult chemistry. Some radical enzymes have unexpectedly been found to harbour a stable radical on an amino acid side‐chain that is distinct from the side‐chains of the active site region; during catalysis, the stable free radical interacts with the active site via radical transfer, and between turnovers it serves as a radical sink.

Key Concepts

  • Radical enzymes utilise single‐electron chemistry to enable difficult chemical reactions.
  • Tyrosyl and tryptophanyl radicals are involved in a large number of biochemical reactions as initiators of catalysis or as partners in radical transfer pathways.
  • Glycyl radicals are oxygen‐sensitive and have only been found in anaerobically expressed enzymes.
  • Transient cysteinyl radicals are important intermediates in a number of radical enzymes.
  • A transient 5′‐deoxyadensyl radical can be formed either by reductive cleavage of the S‐adenosylmethionine cofactor in a radical SAM enzyme or by homolytic cleavage of the vitamin B12 coenzyme AdoCbl.
  • All members of the ribonucleotide reductase enzyme family utilise a transient cysteinyl radical generated by a stable radical in a separate subunit (class I), cleavage of AdoCbl (class II), or a stable glycyl radical (class III).

Keywords: tyrosyl radical; dopa radical; glycyl radical; tryptophanyl radical; cysteinyl radical; 5′‐deoxyadenosyl radical; ribonucleotide reductase

Figure 1. (a) General reaction mechanism of RNRs. (b) General structures of the three RNR classes. (c) Initiator radicals, and intermediates involved in radical transfer and activation. Class I RNR consists of two homodimeric subunits, the catalytic dimer contains the active site and the radical‐containing dimer the stable radical. Class II RNR is monomeric (shown here) or homodimeric (not shown) and harbour an AdoCbl cofactor close to the active site. Class III RNRs are homodimeric with a stable glycyl radical close to the active site; once the glycyl radical is introduced by NrdG, a specific activating enzyme (a radical‐SAM enzyme), the class III RNR can perform multiple turnovers. The catalytic core structure in all three RNR classes is a specific 10‐stranded β/α‐barrel, which also occurs in all glycyl radical enzymes (GREs; see below). Structures used: class I, (human R1: 2WGH, E. coli R2: 1MXR); class II, L. leichmannii monomeric protein (1L1L); class III, bacteriophage T4 (1H7A). The β‐barrel and catalytic radical cysteine (left protomer for dimeric proteins) are oriented in a similar orientation.
Figure 2. (a) Radical transfer pathway in the class Ia holoenzyme (left), and general structure of the R2 protein (right). Structures used: E. coli class Ia R1–R2 complex, (6W4X); E. coli class Ia R2, (1MXR). (b) Structure of the stable radical in the R2 subclasses Ia to Ie. Structures used: Ia, E. coli (1MXR); Ib, C. ammoniagenes (1KGP); Ic, Chlamydia trachomatis (1SYY, 4D8G); Id, Leeuwenhoekiella blandensis (6SF5); Ie, Mesoplasma florum (6GP2). Solvent derived ligands are omitted for clarity. The location of the radical species is indicated in green.
Figure 3. Prostaglandin H synthase. (a) Structure of the active site region and (b) proposed radical reaction mechanism (structure and numbering refer to PGHS‐1 from Ovis aries: 1Q4G). The activation of AA is initiated by site‐selective H‐atom abstraction by a Y· radical. The haem is shown in yellow, and the aspirin analogue 2‐bromoacetoxybenzoic acid and the aspirin antagonist salicylic acid in red. AA, arachidonic acid; PP, protoporphyrin, PGG2, prostaglandin G2; PGH2, prostaglandin H2.
Figure 4. Schematic structure of Photosystem II and the catalytic cycle of the OEC (the Kok cycle). (a) Selected redox‐active components highlighted, including the OEC (CaMn4) and the two tyrosyl radical residues (YD and YD). Also shown is the chromophore P680 and key electron mediators Pheophytin (Pheo) and Quinones A and B. (b) A schematic representation of the Kok cycle, successive light flashes cycles the OEC through four metastable intermediates S0–S3 and the final light flash results in O2 evolution proceeding via the putative S4 state. The primary electron acceptor of the OEC is a tyrosyl radical (Yz) continuously regenerated via oxidation by P680.
Figure 5. The active site core structure of GREs where the Gly‐loop and Cys‐loop meet in a 10‐stranded β/α barrel. Structure used: Bacteriophage T4 class III RNR (1H7A).
Figure 6. Activation, oxygen inactivation, and YfiD‐mediated reactivation of PFL. PFL (blue) with Gly‐loop (red); PFL activating enzyme (green), YfiD (brown). PFL activating enzyme promotes a conformational change of Gly‐loop during activation (1), whereafter active PFL with Gly· is generated (2). Upon oxygen exposure, PFL is irreversibly cleaved at the Gly· site (3), and the C‐terminal PFL peptide is ejected (4). YfiD binds exposed to PFL and is activated by PFL activating enzyme when oxygen has disappeared (5), whereafter Gly· in YfiD occupies the Gly‐loop site in PFL (6).
Figure 7. Schematic comparison of how the 5′‐deoxyadenosyl radical (5′‐dAdo·) is formed from the ‘omega’ intermediate (an organometallic complex of the 5′‐C of the AdoMet‐derived deoxyadenosyl moiety bound to the AdoMet‐coordinating Fe in the [4Fe‐4S] cluster) derived from the radical SAM cofactor (only sulfur of Met residue shown for increased clarity) observed in radical SAM enzymes and from the AdoCbI cofactor (Table ).
Figure 8. The typical TIM barrel in a majority of radical SAM enzymes with AdoMet bound to the [4Fe‐4S] cluster. Structure used: Methanosarcina barkeri str. Fusaro methylornithine synthase (3T7V).
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Högbom, Martin, Sjöberg, Britt‐Marie, and Berggren, Gustav(Sep 2020) Radical Enzymes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0029205]