DOI: 10.1002/cbic.200900777 Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions E. Neil G. Marsh,*[a] Dustin P. Patterson,[a] and Lei Li*[b] 604 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 604 – 621 Adenosine is undoubtedly an ancient biological molecule that confined to a rather narrow repertoire of rearrangement reac- is a component of many enzyme cofactors: ATP, FADH, tions involving 1,2-hydrogen atom migrations; nevertheless, NAD(P)H, and coenzyme A, to name but a few, and, of course, mechanistic insights gained from studying these enzymes have of RNA. Here we present an overview of the role of adenosine proved extremely valuable in understanding how enzymes in its most reactive form: as an organic radical formed either generate and control highly reactive free radical intermediates. by homolytic cleavage of adenosylcobalamin (coenzyme B12, In contrast, there has been a recent explosion in the number AdoCbl) or by single-electron reduction of S-adenosylmethio- of radical-AdoMet enzymes discovered that catalyze a remarka- nine (AdoMet) complexed to an iron–sulfur cluster. Although bly wide range of chemically challenging reactions; here there many of the enzymes we discuss are newly discovered, adeno- is much still to learn about their mechanisms. Although all the sine’s role as a radical cofactor most likely arose very early in radical-AdoMet enzymes so far characterized come from anae- evolution, before the advent of photosynthesis and the pro- robically growing microbes and are very oxygen sensitive, duction of molecular oxygen, which rapidly inactivates many there is tantalizing evidence that some of these enzymes radical enzymes. AdoCbl-dependent enzymes appear to be might be active in aerobic organisms including humans. 1. Introduction 5’-Deoxyadenosyl radical (AdoC) serves two functions in biochem- ical reactions. Firstly, it is an ex- tremely powerful single-electron oxidant that can remove a hy- drogen atom from the least re- active of molecules.[1] This allows cells to catalyze difficult oxida- tion reactions under anaerobic conditions that would otherwise require the oxidizing power of activated oxygen species, as ex- emplified by the oxidations cata- lyzed by cytochrome P450 en- zymes. Secondly, it can function as a catalyst by reversibly ab- stracting a hydrogen atom from the substrate: this allows en- zymes to exploit the reactivity of free radicals to catalyze reactions that would be difficult or impos- sible to effect by ionic chemis- try.[2] As illustrated in Scheme 1, there are two biological mecha- nisms for generating AdoC: ho- molytic cleavage of adenosylco- balamin (coenzyme B12, AdoCbl), which results in cob(II)alamin C and Ado , and single-electron re- Scheme 1. Generation of adenosyl radicals. Top: radical generation by 1-electron reduction of AdoMet complexed duction of S-adenosylmethionine with a [4FeÀ4S] cluster; bottom: radical generation by homolysis of the CoÀC bond of AdoCbl. (AdoMet) complexed to a re- duced iron–sulfur cluster, which C yields Ado , methionine, and the oxidized iron–sulfur cluster. [a] Prof. E. N. G. Marsh, D. P. Patterson Whereas AdoCbl always serves as a cofactor, AdoC generated Department of Chemistry, University of Michigan from AdoMet may be used catalytically as a true cofactor, but Ann Arbor, MI 48109-1055 (USA) more often is consumed as a cosubstrate. E-mail: [email protected] There have been significant advances in understanding the [b] Prof. L. Li Department of Chemistry and Chemical Biology, Indiana University mechanisms and biological roles of this group of enzymes in Purdue University Indianapolis, Indianapolis, IN 46202 (USA) the last few years, especially the discovery of many new radi- E-mail: [email protected] ChemBioChem 2010, 11, 604 – 621 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 605 E. N. G. Marsh, L. Li et al. cal-AdoMet enzymes. Here we present an overview of adenosyl that discuss various aspects of adenosyl radical biochemistry in radical enzymes, in particular contrasting radical-AdoMet and more detail than space permits here.[1–15] AdoCbl-dependent enzymes, and discuss whether radical- The first member of this class of enzymes to be identified AdoMet enzymes might be active in animals as well as anaero- was glutamate mutase, an AdoCbl-dependent enzyme in- bic microbes. We also refer the reader to several recent reviews volved 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 bio- chemical function for vitamin B12, which is the precursor to Neil Marsh received his undergraduate AdoCbl. In humans the requirement for vitamin B12 derives, in education at Christ’s College, Cam- part, from the AdoCbl-dependent enzyme methylmalonyl-CoA bridge University. He remained at mutase, which is involved in odd-chain fatty acid metabo- Cambridge for his Ph.D. studies under lism.[14, 20] The radical nature of these reactions was first postu- the guidance of Prof. Peter Leadlay in lated by Abeles through work on dioldehydratase.[21,22] The first the Department of Biochemistry and radical-AdoMet enzyme, lysine-2,3-aminomutase (LAM), was then studied as a post-doctoral fellow not discovered until 20 years later,[23] also by Barker, who with Prof. Craig Townsend at Johns noted the similarity of the reaction to the rearrangements cata- Hopkins University, Baltimore. He re- lyzed by AdoCbl-dependent aminomutases. Another 20 years turned to Cambridge to initiate his would pass before the second radical-AdoMet enzyme would own research program as a Royal Soci- be discovered through studies in J. Knappe’s laboratory on ety University Research Fellow. In 1995 pyruvate formate-lyase (PFL),[24] an enzyme that converts pyru- he moved to the University of Michigan where he is currently Pro- vate to formate and acetyl-CoA as part of the anaerobic fessor of Chemistry and Biological Chemistry. His research interests metabolism of glucose in E. coli. PFL was the first-discovered include the mechanisms by which enzymes generate and control member of a group of enzymes that contain a radical centered free radicals and the use of fluorinated amino acids to modulate on the a-carbon of the specific protein glycyl residue that is the structure, stability and biological activity of proteins and pep- required in the catalytic mechanism. Knappe and colleagues tides. showed that the glycyl radical in PFL is installed by a specific activase enzyme that uses an adenosyl radical, derived from Lei Li received his Ph.D. from Johns AdoMet, to abstract a hydrogen atom from gly734 of PFL.[25] Hopkins University under the guidance Since then studies in numerous laboratories have identified of Prof. Kenneth D. Karlin in 2005, radical-AdoMet enzymes that participate in a remarkably wide studying copper–dioxygen-complex- range of chemical transformations; representative examples mediated aliphatic CÀH bond and that are discussed in this review are summarized in Table 1. DNA nucleobase oxidation. He then These efforts were greatly aided by a sequence comparison moved to Prof. Neil Marsh’s laboratory study, published in 2001 that, based on the CX3CX2C motif at the University of Michigan, investi- shared by the known radical-AdoMet enzymes, identified a fur- gating the reaction mechanism of ben- ther 600 enzymes that can utilize AdoMet in this manner[26] zylsuccinate synthase, a glycyl radical (today the sequence database contains around 3000 putative enzyme involved in toluene degrada- radical-AdoMet enzymes[5]). Although some of the sequences tion. In 2008, he was the recipient of were homologues of known radical-AdoMet enzymes from dif- an NIH Pathway to the Independence Award. In 2009, he started ferent bacteria, and many more sequences were from com- his independent career as an assistant professor in the Department pletely unknown proteins, some were from known proteins for of Chemistry at Indiana University-Purdue University Indianapolis. which a connection to radical-AdoMet chemistry had not been His research focuses on the mechanistic elucidation of oligonucleo- made. Here the sequence motif provided the clue needed to tide modifications mediated by radical-AdoMet enzymes. guide biochemical studies elucidating the role of AdoMet in the enzyme reactions. Recent findings suggest that the radical- Dustin Patterson received his BS in AdoMet family could be even larger: in 2006, an elongator Chemistry with a specialization in Bio- subunit Elp3 from Methanocaldococcus jannaschii was found chemistry from Bowling Green State to contain a [4FeÀ4S] cluster coordinated by a CX4CX2C se- University, Ohio. He is currently a grad- quence. This cluster is able to bind AdoMet and generate 5’- uate student at the University of Michi- deoxyadenosine probably via a similar sulfur–carbon bond- gan working in Prof. Neil Marsh’s re- cleavage chemistry.[27] Additional evidence has been obtained search group. His research projects in- on ThiC, which also contains a CX4CX2C motif, that reductive clude characterizing the structure of cleavage of AdoMet to generate AdoC is a feature of the mech- the glycyl radical enzyme benzylsucci- [28,29] anism. These findings indicate that the CX3CX2C motif nate synthase and the development of might not be the definitive sequence for this enzyme super- new strategies to assemble supra- family. Furthermore, the Elp3 subunits from Saccharomyces cer- molecular protein complexes. evisiae, Schizosaccaromyces pombe, and human were found to 606 www.chembiochem.org 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 604 – 621 Adenosyl Radical enzymes are not especially Table 1. Summary of AdoCbl and radical-AdoMet-dependent enzymes discussed in this review. oxygen sensitive, all radical- Enzyme Function Ref. AdoMet enzymes studied to Adensylcobalamin-dependent radical enzymes date must be handled under rig- lysine-5,6-aminomutase aminomutase [2], [36] orously anaerobic conditions to ornithine-4,5-aminomutase aminomutase [2] maintain their activity.