Adenosyl radical: reagent and catalyst in enzyme reactions.

5′-deoxyadenosyl radical (Ado•) serves two functions in biochemical reactions. Firstly, it is an extremely powerful single-electron oxidant that can remove a hydrogen atom from the least reactive of molecules.[1] This allows cells to catalyze difficult oxidation reactions under anaerobic conditions that would otherwise require the oxidizing power of activated oxygen species, as exemplified by the oxidations catalyzed by cytochrome P450 enzymes. Secondly, it can function as a catalyst by reversibly abstracting a hydrogen atom from the substrate: this allows enzymes to exploit the reactivity of free radicals to catalyze reactions that would be difficult or impossible to effect by ionic chemistry.[2] As illustrated in figure 1, there are two biological mechanisms for generating Ado•: homolytic cleavage of adenosylcobalamin (coenzyme B12, AdoCbl), which results in cob(II)alamin and Ado•, and single-electron reduction of S-adenosylmethionine (AdoMet) complexed to a reduced iron-sulfur cluster, which yields Ado•, methionine and the oxidized iron-sulfur cluster. Whereas AdoCbl is always serves as a cofactor, Ado• generated from AdoMet may be used catalytically as a true cofactor, but more often is consumed as a co-substrate. Figure 1 Generation of adenosyl radicals. Top: radical generation by 1-electron reduction of AdoMet complexed with a [4Fe-4S] cluster; bottom: radical generation by homolysis of the Co-C bond of AdoCbl. There have been significant advances in understanding the mechanism and biological roles of this group of enzymes in the last few years, especially the discovery of many new radical AdoMet enzymes. Here we present an overview of adenosyl radical enzymes, in particular contrasting radical AdoMet and AdoCbl-dependent enzymes, and discuss whether radical AdoMet enzymes may be active in animals as well as anaerobic microbes. We also refer the reader to several recent reviews that discuss various aspects of adenosyl radical biochemistry in more detail than space permits here.[1–15] The first member of this class of enzymes to be identified was glutamate mutase, an AdoCbl-dependent enzyme involved in the fermentation of glutamate by various bacteria, which was discovered by H.A. Barker and colleagues in the late 1950s.[16–19] Notably, this discovery provided a specific biochemical function for vitamin B12, which is the precursor to AdoCbl. In humans the requirement for vitamin B12 derives, in part, from the AdoCbl-dependent enzyme methylmalonyl-CoA mutase which is involved in odd-chain fatty acid metabolism.[14, 20] The radical nature of these reactions was first postulated by Abeles through work on dioldehydratase.[21, 22] The first radical-AdoMet enzyme, lysine-2,3-aminomutase (LAM), was not discovered until 20 years later,[23] also by Barker, who noted the similarity of the reaction to the rearrangements catalyzed by AdoCbl dependent aminomutases. Another 20 years would pass before the second radical-AdoMet enzyme would be discovered through studies in J. Knappe’s laboratory on pyruvate formate-lyase (PFL),[24] an enzyme that converts pyruvate to formate and acetyl-CoA as part of the anaerobic metabolism of glucose in E. coli. PFL was the first-discovered member of a group of enzymes that contain a radical centered on the α-carbon of specific protein glycyl residue that is required in the catalytic mechanism. Knappe and colleagues showed that the glycyl radical in PFL is installed by a specific activase enzyme that uses an adenosyl radical, derived from AdoMet, to abstract a hydrogen atom from gly-734 of PFL.[25] Since then studies in numerous laboratories have identified radical-AdoMet enzymes that participate in a remarkably wide range of chemical transformations; representative examples that are discussed in this review are summarized in Table 1. These efforts were greatly aided by a sequence comparison study, published in 2001, that, based on the CX3CX2C motif shared by the known radical-AdoMet enzymes, identified a further 600 enzymes that may utilize AdoMet in this manner[26] (today the sequence data base contains around 3000 putative radical-AdoMet enzymes[5]). Although some of the sequences were homologs of known radical-AdoMet enzymes from different bacteria, and many more sequences were from completely unknown proteins, some were from known proteins for which a connection to radical-AdoMet chemistry had not been made. Here the sequence motif provided the clue needed to guide biochemical studies elucidating the role of AdoMet in the enzyme reactions. Recent findings suggest that the radical AdoMet family could be even larger: in 2006, an elongator subunit Elp3 from Methanocaldococcus jannaschii was found to contain a [4Fe-4S] cluster coordinated by a CX4CX2C sequence. This cluster is able to bind AdoMet and generate 5′-deoxyadenosine probably via a similar sulfur-carbon bond cleavage chemistry.[27] Additional evidence has been obtained on ThiC, which contains a CX4CX2C motif as well, that the Ado• radical is generated via reductive cleavage of AdoMet.[28, 29] These findings indicate that the CX3CX2C motif may not be the definitive sequence for this enzyme superfamily. Furthermore, the Elp3 subunits from Saccharomyces cerevisiae, Schizosaccaromyces pombe and human were found to possess a CX9CX2C motif.[27] This suggests that more, as yet unrecognized enzymes, may utilize this radical AdoMet chemistry. In contrast, AdoCbl-dependent enzymes appear to be relatively rare; only 10 are known so far,[12] with no new enzymes discovered in over 20 years. Their reactions are restricted to either isomerization or elimination reactions. Although a B12-binding motif has been described,[30, 31] it is not shared by all enzymes, nor is it unique to AdoCbl enzymes because it also occurs in some cobalamin-dependent methyl transferases; this has made identifying new AdoCbl enzymes in sequence databases difficult. Table 1 Summary of AdoCbl and radical AdoMet dependent enzymes discussed in this review 2. Similarities and differences between radical AdoMet and AdoCbl enzymes The most significant difference between AdoCbl and radical AdoMet enzymes is their oxygen sensitivity. Whereas AdoCbl enzymes are not especially oxygen sensitive, all radical AdoMet enzymes studied to date must be handled under rigorously anaerobic conditions to maintain their activity. Reactive oxygen species rapidly oxidize and destroy the iron-sulfur clusters in these enzymes. Oxygen sensitivity may have provided the pressure for the evolution of AdoCbl-dependent enzymes; however, as discussed below, radical AdoMet enzymes may be stable in the microenvironment of a cell even under aerobic conditions. Although a detailed discussion of their structures is beyond the scope of this review, both classes of enzyme are built on variations of the β/α barrel (TIM barrel) scaffold. The structures of representative members of the two classes of enzymes are compared in figure 2. All AdoCbl enzymes for which structures are known comprise “complete” barrels – 8 stranded barrels for methylmalonyl-CoA mutase,[32] diol dehydratase,[33] glutamate mutase [34] and lysine-5,6-aminomutase,[35] and a 10-stranded barrel for ribonucleotide reductase.[36] Radical AdoMet enzymes exhibit more structural diversity: the catalytic domains of biotin synthase, ThiC and HydE are complete 8-stranded barrels [29, 37, 38] whereas HemN, LAM, MoaA and PFL activase are 6-stranded (β/α)6 “3/4” barrels [39–42] in which the fold is opened out to facilitate entry of the substrate.[3, 5] This opening out is most marked for PFL-activase, which must accommodate a protein substrate within its active site.[41] Figure 2 Comparison of the structures of AdoCbl and radical AdoMet enzymes. Left: the structures of biotin synthase ((β/α)8 complete barrel), glutamate mutase ((β/α)8 complete barrel) and lysine-2,3-aminomutase ((β/α) ... Lastly we note that, intriguingly, both AdoMet and cobalamin, as methylCbl (MeCbl), also function as methyl donors, indeed in MeCbl-dependent methionine synthase AdoMet can act as a methyl donor to cob(II)alamin under some conditions.[12] In both classes of enzymes methyl transfer involves ionic reactions, rather than radical chemistry; in the cobalamin-dependent methylases cobalt cycles between +3 (methylated) and +1 (unmethylated) oxidation states. This illustrates a further similarity between the intrinsic reactivity of sulfonium ions and organo-cobalt complexes that has been exploited by these two classes of enzymes.